We have determined whether TM3 cells, a cell line derived from a murine Leydig cell tumor, (a) transcribe messenger RNAs encoding the beta A-subunits of inhibin/activin, and activin receptors I, II, and IIB, and (b) produce activin-A protein. Messenger RNAs for inhibin/activin beta A-subunits and activin receptors II, and IIB in TM3 cells were localized and expressed using in situ hybridization and the reverse transcription-polymerase chain reaction (RT-PCR) analysis, respectively. The identify of the RT-PCR products was confirmed by DNA sequencing of PCR products. Immunocytochemically, activin-A was localized in these cells. We observed that messenger RNAs encoding activin beta A-subunit as well as activin receptors II, and IIB were expressed and activin protein was produced by TM3 cells. These findings suggest that activin-A may have autocrine functions in TM3 cells and that activin-A may be involved in regulating the growth and differentiation of mouse Leydig cells.