Neutral-carrier pH- and Ca-sensitive microelectrodes were used to investigate the relationship between junctional electrical resistance and either pHi or [Ca2+]i in crayfish septate axons uncoupled by acidification. For measuring [Ca2+]i a new neutral carrier sensor sensitive to picomolar [Ca2+] and virtually insensitive to other ions was used. Uncoupling was induced by superfusing the axons with Na-acetate solutions (pH 6.3). With acetate, the time course of changes in junctional resistance differed markedly from that of pHi or [H+]i, and [H+]i peaked 40-90 sec before junctional resistance. The difference in shape and peak time between pHi and junctional resistance curves caused significant hysteresis in the pHi versus junctional resistance relationship. In addition, junctional resistance maxima reached with slow acidification rates were 3-4 times greater than those with fast acidification of similar magnitude. With acetate, [Ca2+]i increased by approximately one order of magnitude from basal values of 0.1-0.3 microM. The curves describing the time course of changes in [Ca2+]i and junctional resistance matched well with each other in shape, peak time and magnitude. Both junctional resistance and [Ca2+]i recovered following a single exponential decay with a time constant of approximately 2 min. Different rates of acidification caused increases in [Ca2+]i and junctional resistance comparable in magnitude. The data indicate that the increase in junctional resistance induced by acidification is more closely related to [Ca2+]i than to [H+]i.