The siphon withdrawal reflex of Aplysia undergoes differential classical conditioning with cutaneous stimulation of the siphon or mantle shelf as the discriminative conditioned stimuli (CS+ and CS-) and shock to the tail as the unconditioned stimulus (US). The reflex has proved to be useful for analyzing the neural mechanisms of conditioning. To test the generality of this experimental system, we have begun to compare the properties of conditioning in Aplysia with those of conditioning in vertebrates. We first examined the effect of the interstimulus interval (ISI) by varying the time between presentation of the CS+ and the US in different groups of animals. Significant differential conditioning was obtained when the onset of the CS+ preceded the onset of the US by 0.5 sec, and marginal conditioning was obtained when the ISI was 1.0 sec. By contrast, no significant conditioning occurred when the CS+ preceded the US by 2, 5, or 10 sec, when the onsets of the stimuli were simultaneous, or when US onset preceded the CS+ by 0.5, 1.0, or 1.5 sec (backward conditioning). We next examined the effect of contingency by giving one group of animals normal differential conditioning, and a second group the same training but with additional USs inserted between the paired trials. Presentation of these additional USs reduced the degree to which the US was contingent on the CS+, but did not change the number of pairings. Animals receiving normal training again showed significant conditioning, whereas animals receiving additional USs showed no conditioning.(ABSTRACT TRUNCATED AT 250 WORDS)