The mammalian tachykinin peptides, substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) are encoded by distinct mRNAs derived from separate preprotachykinin (PPT) genes. The SP/NKA-encoding PPT gene generates three mRNAs by alternative RNA processing: α-PPT mRNA, which encodes SP only, and β- and γ-PPT mRNAs, which encode both SP and NKA. The NKB-encoding PPT gene generates mRNAs that produce NKB. The distribution and cellular localization of SP, NKA and NKB mRNAs in the rat retina were studied by RNA blot and in situ hybridization techniques. Blot hybridization analysis of retinal RNA extracts with [32P]-labeled RNA probes complementary to SP/NKA and NKB mRNAs demonstrated single bands of hybridization at 1300 and 900 bases, respectively. Solution hybridization-nuclease protection experiments showed multiple SP/NKA-encoding transcripts with relative levels of ρ-PPT mRNA > β-PPT mRNA ≫ α-PPT mRNA. In situ hybridization histochemistry with [35S]-labeled antisense RNAs demonstrated thatSP/NKA-encoding transcripts are expressed in small-to-medium somata located in the proximal inner nuclear, inner plexiform, and ganglion cell layers, whereas NKB-encoding transcripts are expressed in small-to-medium somata located only in the ganglion cell layer. In this layer, cells containing NKB mRNAs are more numerous than those containing SP/NKA mRNAs. Only background labeling was observed in sections incubated with sense RNA probes, pretreated with RNase A prior to hybridization or incubated in hybridization buffer without the labeled probe. Immunohistochemical studies with a monoclonal antibody directed to the conserved COOH-terminal sequence of the tachykinin peptides revealed tachykinin-like immunoreactive somata with similar size and distribution to those containing SP/NKA- and NKB-encoding transcripts. These results indicate that both SP/NKA and NKB mRNAs are present in the rat retina and that the PPT genes are differentially expressed in specific cell populations. The size and distribution of these cells suggest that they are amacrine and displaced amacrine cells, however, the possibility that tachykinins are present also in ganglion cells in the rat retina cannot be ruled out.