Three experiments investigated conditioning of the rabbit’s nictitating membrane response (NMR) in a second-order conditioning procedure which intermixed first-order trials (CS1-US) and second-order trials (CS2-CS1) from the outset of training. Experiment 1 provided a controlled demonstration that substantial levels of second-order conditioning can be obtained with the NMR preparation. Experiment 2 showed that the level of CR acquisition to CS2 was an inverse function of the CS2-CS1 interval over the values of 400, 800, and 2,400 msec. Experiment 3 found that CR acquisition to CS2 and CS1 in second-order conditioning varied in a parallel fashion across CS1-US intervals. Similarly, Experiment 3A found that the level of CR acquisition to the two components of a serial compound (CSA-CSB-US) varied in a parallel fashion as a function of the CSB-US interval. The results of the CS2-CS1 and CS1-US interval manipulations were all predictable from the known CS-US interval effects in NMR conditioning with a single CS. The present results are discussed with regard to their implications for accounts of serial compound conditioning and second-order conditioning.