In first-order Pavlovian conditioning, learning is acquired by pairing a conditioned stimulus (CS) with an intrinsically motivating unconditioned stimulus (US; e.g., food or shock). In higher-order Pavlovian conditioning (sensory preconditioning and second-order conditioning), the CS is paired with a stimulus that has motivational value that is acquired rather than intrinsic. This review describes some of the ways higher-order conditioning paradigms can be used to elucidate substrates of learning and memory, primarily focusing on fear conditioning. First-order conditioning, second-order conditioning, and sensory preconditioning allow for the controlled demonstration of three distinct forms of memory, the neural substrates of which can thus be analyzed. Higher-order conditioning phenomena allow one to distinguish more precisely between processes involved in transmission of sensory or motor information and processes involved in the plasticity underlying learning. Finally, higher-order conditioning paradigms may also allow one to distinguish between processes involved in behavioral expression of memory retrieval versus processes involved in memory retrieval itself.By reducing learning to its most rudimentary components, the influence of undefined and uncontrollable confounding variables can be minimized. Consequently, much of the progress that has been achieved in searching for the neural substrates of learning and memory has been made using some of the simplest forms of learning. In one such paradigm, first-order Pavlovian conditioning, a conditioned stimulus (CS, such as a tone or light) acquires motivational significance by being paired with an intrinsically aversive or rewarding unconditioned stimulus (US, such as foot shock or food). Learning is evaluated by the ability of the CS to elicit a conditioned response (CR) in anticipation of the occurrence of the US. The use of first-order conditioning has revealed genetic and cellular mechanisms underlying learning and memory in species ranging from the fruit fly and sea snail to the mouse and rat.Thus far, less attention has been paid by neurobiologists to the potential uses of higher-order Pavlovian conditioning, learning phenomena in which a CS (S2) acquires associative strength by being paired with another CS (S1) rather than with a US. The pairing of S2 with S1 may occur before S1 is paired with the US (sensory preconditioning) or after S1 has been paired with the US (second-order conditioning; see Table 1). The cardinal feature of both sensory preconditioning and second-order conditioning-and that which recommends these paradigms to the service of neurobiologists interested in learning and memory-is that S2 acquires associative strength even though it is never paired directly with a US. This article will describe two promising avenues of research in higher-order conditioning. First, the fact that reinforcing value is acquired makes higher-order conditioning well suited to investigating the neural substrates of different forms of reinforcement. Second, the abs...