1. By use of a 'grease-gap' technique, the depolarizing effects of adenosine 5'-triphosphate (ATP) and ATP analogues on the rat isolated vagus nerve were determined in normal and in Ca2+/Mg(2+)-free (+ 1 x 10(-3) M ethylenediamine tetraacetic acid) physiological salt solution (PSS). 2. In normal PSS, ATP produced concentration-dependent depolarization responses but the concentration-effect curve to ATP was incomplete and a maximum effect was not achieved. The threshold concentration for depolarization was 1 x 10(-5) M and at the highest concentration tested (1 x 10(-3) M) the peak amplitude of the response to ATP only amounted to 71% of the depolarization produced by a near maximal response to 5-hydroxytryptamine (5-HT, 1 x 10(-5) M). 3. In Ca2+/Mg(2+)-free PSS, ATP produced depolarization responses at much lower concentrations and of markedly larger amplitude. Under these conditions, the threshold concentration for depolarization was 1-3 x 10(-7) M and the maximal response to ATP amounted to 526% of the response to 5-HT (1 x 10(-5) M) in normal PSS. The concentration-effect curve to ATP was sigmoid, with a defined maximum effect and a mean EC50 value of 1.2 x 10(-6) M. 4. In contrast to the effects on responses to ATP, the absence of divalent cations in the PSS did not modify the effective concentrations of either alpha, beta-methylene ATP or 5-HT. However, the maximum responses to both alpha, beta-methylene ATP and 5-HT were significantly increased in Ca2+/Mg(2+)-free PSS. 5. The depolarizing effects of several analogues of ATP were determined in Ca2+/Mg2+-free PSS.ATP-gamma-S and 2-methylthioATP were of similar potency to ATP (respective equi-effective molar ratios(EMRs) of 1.9 and 1.3, where ATP = 1) and similar maximum responses were obtained. Alpha, beta-MethyleneATP, beta, gamma-methylene ATP and ,beta, gamma-imido ATP were considerably less potent than ATP, analysis yielding mean EMRs of 48.9, 85.0 and 60.0, respectively. Maximum responses to these latter three agonists were not obtained at the highest concentrations tested (1 x 10-4-3 X 10- M). Benzoyl ATP, adenosine 5'-0-(2-thiodiphosphate) and adenosine diphosphate produced only small depolarizing responses at high concentrations (>1 x 10-4 M). Adenosine monophosphate, adenosine and uridine S'-triphosphate each had little or no depolarizing effect in Ca2+/Mg2+-free PSS.6. These data demonstrate that in the absence of divalent cations the excitatory actions of some, but not all, purine nucleotides in the rat vagus nerve are markedly potentiated. In Ca2+/Mg2+-free PSS, the rank order of agonist potencies was ATP = 2-methylthioATP = ATP-gamma-S>> alpha,beta-methylene ATP = beta, gamma imido ATP = P,y-methylene ATP. These findings are in stark contrast to our previous observations in normal PSS where the rank order of agonist potencies for these nucleotides was alpha,beta-methyleneATP> ATP-gamma-S > beta,gamma-imido ATP = beta,gamma-methylene ATP> 2-methylthioATP> ATP.7. We suggest that the two different rank orders of potency can be explained by differential metabolism involving Ca2+/Mg2+-dependent ectonucleotidases. If so, these data indicate that ATP and 2-methylthioATP are inherently more potent than alpha,beta-methylene ATP as agonists at neuronal P2X purinoceptors in the rat vagus nerve. The possible implications of these findings to the present system for subclassifying P2 purinoceptors are profound.