Using degenerate primers designed to amplify genes containing homeodomains, we have used reverse transcription and polymerase chain reaction to amplify and clone a rat homeobox gene. Based on the nucleotide and predicted amino acid sequences, the rat cDNA clone contains a high degree of sequence similarity to murine genes which are members of the paired-like class of homeobox genes (Ptx2, Otlx2, solurshin and Ptx1). Considering the high degree of sequence similarity and similar restricted expression patterns, we have named the cloned rat gene rPtx2 (rat Ptx2 homolog). Northern analysis revealed two rPtx2 transcripts expressed in the developing rat brain. Yet, only a single gene was detected by Southern blot hybridization, suggesting that multiple messages are the result of alternative transcriptional initiation, splicing or processing of a common message. The expression pattern of rPtx2 was further delineated by in situ hybridization to rat embryos. Within the brain, tissue specific expression was observed in the differentiating neural cells of the posterior hypothalamus, tegmentum, and rhombomere r1. Expression was also observed in the developing pituitary, maxilla, mandible, tongue and umbilical cord. To further study the control of Ptx2 gene expression, we used an in vitro model for neural differentiation by treating mouse embryonic stem cells with retinoic acid. Within 24 h and prior to detection of a neural phenotype in the culture, murine Ptx transcripts were induced and remained elevated for at least 6 days. This suggests that retinoic acid may be an important inductive signal which regulates the developmental and tissue-specific expression of Ptx2.