The tachykinin neuropeptides, substance P and substance K, are produced in nociceptive primary sensory neurons and in many brain regions involved in pain signaling. However, the precise role and importance of these neuropeptides in pain responses has been debated. We now show that mice that cannot produce these peptides display no significant pain responses following formalin injection and have an increased pain threshold in the hotplate test. On the other hand, the mutant mice react normally in the tail f lick assay and acetic acid-induced writhing tests. These results demonstrate that substance P and͞or substance K have essential functions in specific responses to pain.The tachykinins are a family of structurally related neuropeptides. In the mouse, they are encoded by the genes Tac1 and Tac2. Tac1 produces substance P, substance P (neurokinin A), neurokinin A (3-10), neuropeptide K, and neuropeptide ␥ as a result of differential splicing and posttranslational processing (1-4). Tac2 produces the peptide neurokinin B.The undecapeptide substance P was first detected by von Euler and Gaddum (5) in 1931. Its structure was revealed by Leeman and her coworkers (6, 7) in 1971. The Tac1 cDNA was cloned in 1983 by Nakanishi and his coworkers (2,8). The Tac1 gene is expressed in many regions in the central and peripheral nervous system, as well as in nonneuronal tissues. Substance P has been implicated in a variety of physiological processes including cardiovascular, respiratory, and gastrointestinal functions; inflammatory responses; and nociception. In addition, Hunt and coworkers have suggested that substance P may be involved in axon guidance during embryonic development (9).The precise role of substance P in these processes is unclear. For example, substance P is synthesized in nociceptive primary sensory neurons, which send C-and A␦ fibers to dorsal horn projection neurons in lamina I and IV-V, and to nociceptionspecific interneurons in lamina II-III of the spinal cord. Axons of projection neurons terminate in many supraspinal nuclei that are involved in pain transmission (10, 11). Nociceptive stimulation triggers the release of substance P from C-afferent terminals in the marginal layers of the spinal cord (12), evokes slow excitatory postsynaptic potentials in second-order sensory neurons in the dorsal horn, and facilitates their activation (13). These data, together with other functional evidence (11,14), indicated an important role for substance P in the processing of nociceptive signals.We have begun to use a genetic approach to study the functions of tachykinin peptides. As a first step, we have generated mice with a targeted mutation in the Tac1 gene. These mice are viable and fertile, but exhibit striking defects in nociceptive behaviors. MATERIALS AND METHODS Generation and Breeding of Tac1؊͞؊ Mice. Tac1 mutations were established by homologous recombination in MPI2 embryonic stem (ES) cells according to standard protocols (15). One mutant ES cell line was used to derive chimeras by morula aggrega...