Thy-1 is a well characterized glycoprotein known to be variably expressed on the surface of different cell types. Serological analysis of a limited number of teratocarcinoma-derived cell lines suggested that mouse embryonal carcinoma cells do not express Thy-1 and that its expression is associated with the appearance of differentiated cells. In this report we show that monoclonal antibody 1aG4, recognizing Thy-1.2 epitope, binds specifically to P19 embryonal carcinoma cells and their undifferentiated subclones. A number of control experiments confirmed that 1aG4 antibody binds to the Thy-1.2 glycoprotein expressed on the surface of P19 embryonal carcinoma cells and not to the antigen expressed on differentiated derivatives of these cells or to a cross-reactive epitope. Transcriptional activity of the Thy-1 gene in undifferentiated P19 cells was shown by transfection experiments in which transfer of the Thy-1.1 gene into P19 cells resulted in stable expression of the Thy-1.1 antigen on the surface of recipient cells. Direct evidence for the presence of Thy 1 mRNA in P19 cells was obtained by Northern blot analysis with a Thy-1-specific cDNA probe. Treatment of P19 cells with retinoic acid resulted in a decrease in the expression of Thy-1 antigen which preceded changes in morphology of the cells. These data indicate that Thy-1 is a developmentally regulated surface marker of P19 embryonal carcinoma cells which is amenable to direct genetic analysis.