Expression of mRNA in monolayer cells transfected with recombinant DNA was demonstrated by in situ hybridization with biotin- or (32)P-labeled probes. The biotin-labeled probe and hybrid detection system used in this study could detect 1 pg of target DNA and a single copy gene in mammalian cells using filter hybridization techniques. Several conditions of in situ hybridization were optimized with neomycin resistant (neo(r)) cells, which constitutively expressed neo(r) mRNA derived from pSV2neo. The proteolytic digestion was found to be the most critical procedure. The in situ hybridization demonstrated that neo(r) mRNA was localized in the cytoplasm of the neo(r) cells. Cf2Th cells derived from a canine fetal thymus cell were transfected with the cloned provirus genome of a retrovirus produced in a human lymphoblastoid cell line, and transient expressions of viral RNA and antigens were monitored by in situ hybridization and immunoperoxidase staining, respectively. About 48 hours after transfection, several percent of the transfected cells were positive for expression of both viral RNA and antigens by these cytochemical methods. These observations indicate that in situ hybridization is a useful method for detecting the expression of the gene introduced into mammalian cells.