Second-order conditioning was examined using the rabbit eyeblink paradigm and the gerbil CER paradigm. Pavlov’s hypothesis that stimulus overlap on second-order trials produces conditioned inhibition and that nonoverlap leads to second-order conditioning was not confirmed. Our results also revealed that the manner in which first-order and second-order trials are intermixed has an important influence on the properties of the second-order CS. A within-session mixture of first- and second-order trials tended to produce second-order conditioning, and a between-session mixture tended to produce conditioned inhibition. Second-order conditioning was more prominent with the gerbil fear response than with the rabbit eyelid response.