Effects of forward and backward conditioned-unconditioned stimulus (CS-US) intervals on classical conditioning of the flexion reflex were examined in a cat spinal preparation. Interstimulus intervals (ISIs) ranging from +3.0 to -3.0 sec were employed in 9 experimental groups and the results compared with those of an explicitly unpaired control group. Forward conditioning produced an asymmetrical, inverted U-shaped gradient relating magnitude of conditioning to ISI for both acquisition and extinction. The optimum ISI was 1.0 sec. Backward ISIs also produced excitatory conditioning, with optimal conditioning at -0.25 sec. Unlike forward conditioning, backward conditioning produced little sign of retention during extinction trials. The results, which parallel in several ways those of ISI effects in studies of intact animals, support the hypothesis that backward and forward conditioning may be fundamentally different phenomena, under the control of different neural processes.