In the nervous system of the hawkmoth, Manduca sexta, cells expressing the period (per)gene were mapped by in situ hybridization and immunocytochemical methods. Digoxigenin-labeled riboprobes were transcribed from a 1-kb M. sexta per cDNA. Monoclonal anti-PER antibodies were raised to peptide antigens translated from both M. sexta and Drosophila melanogaster per cDNAs. These reagents revealed a widespread distribution of per gene products in M. sexta eyes, optic lobes, brains, and retrocerebral complexes. Labeling for per mRNA was prominent in photoreceptors and in glial cells throughout the brain, and in a cluster of 100-200 neurons adjacent to the accessory medulla of the optic lobes. Daily rhythms of per mRNA levels were detected only in glial cells. PER-like immunoreactivity was observed in nuclei of most neurons and glial cells and in many photoreceptor nuclei. Four neurosecretory cells in the pars lateralis of each brain hemisphere exhibited both nuclear and cytoplasmic staining with anti-PER antibodies. These cells were positively identified as Ia(1) neurosecretory cells that express corazonin immunoreactivity. Anti-corazonin labeled their projections in the brain and their neurohemal endings in the corpora cardiaca and corpora allata. Four pairs of PER-expressing neurosecretory cells previously described in the silkmoth, Anthereae pernyi, are likely to be homologous to these PER/corazonin-expressing Ia(1) cells of M. sexta. Other findings, such as widespread nuclear localization of M. sexta PER and rhythmic expression in glial cells, are reminiscent of the period gene of D. melanogaster, suggesting that some functions of per may be conserved in this lepidopteran species.