Effects of stimulation of intramural nerves in the circular smooth muscle layer of the body of the oesophagus of the opossum (Didelphis marsupialis) were studied, simultaneously measuring the membrane potential of muscle cells using the sucrose-gap technique and contractions of the muscle. Electrical field stimulation of the preparation, superfused with Krebs solution at 27 degrees C, induced a transient hyperpolarization of the smooth muscle cell membrane (i.j.p.) followed by a transient depolarization on which muscle action potentials were often superimposed. The muscle did not develop active tension spontaneously; it therefore did not relax during the i.j.p., but often contracted during the 'off' depolarization. The i.j.p. and the responses following it were characterized as mediated by intramural, non-adrenergic, non-cholinergic (n.a.n.c.) nerves. The i.j.p. amplitude was reduced by raising the external K concentration. When Cl was replaced by isethionate, a small hyperpolarization of the smooth muscle cell membrane ensued along with a comparable small reduction of the i.j.p. amplitude. The 'off' activity following the i.j.p. disappeared completely in Cl-free medium. Apamin (10(-7)-10(-5) M) did not influence this preparation nor the i.j.p. Adenosine and its related adenine nucleotides in concentrations up to 10(-3) M hardly affected the preparation. Prolonged superfusion with adenosine and 6-chloroadenosine revealed a gradually increasing attenuation of the i.j.p. Exogenously applied vasoactive intestinal polypeptide (VIP) induced rhythmic depolarizations of the smooth muscle cell membrane and spontaneous contractions, which were insensitive to neurotoxins but absent in Cl-free media. Field stimulation in the presence of VIP caused an i.j.p. which transiently interrupted the VIP-induced contractile responses. It is concluded that the inhibitory mediator of the intramural n.a.n.c. nerves present in the circular smooth muscle layer of the opossum oesophagus is neither a purine nor VIP. The i.j.p. may result from a selective increase in K permeability of the smooth muscle cell membrane, the 'off' depolarization may involve an increase in the Cl ion conductance. The suggestion is made that the release of neurotransmitter from intramural n.a.n.c. nerves is modulated presynaptically via P1-purinoceptors and that VIP is a likely candidate for an excitatory transmitter, released simultaneously with the inhibitory transmitter.