This study demonstrates that individual animals can concurrently acquire differently timed conditioned eyelid responses using a differential conditioning procedure in which distinctive conditioned stimuli (CSs) are individually paired with an unconditioned stimulus, with each using a different interstimulus interval (ISI). This promotes robust conditioning, and the timing of the conditioned responses is appropriate for the respective ISIs, differs for each CS to the extent that the ISIs are dissimilar, and is apparent in individual trials. This procedure was used to demonstrate that response timing is not a function of associative strength. These data suggest response timing is mediated by an ability to make temporal discriminations during the CS. The within-animals comparisons made possible by this differential conditioning should facilitate lesion and unit recording analyses of the neural basis of response timing.