The potent tumor promoter 12-0-tetradecanoyl phorbol 13-acetate (TPA) and phenobarbital (PB) were tested for their abilities to promote effect of these agents on the in vitro malignant transformation of the fetal rat brain cells exposed in utero to Ethylnitrosourea (ENU). Rat fetal brain cells were transferred to cultured system at 72 hours after single pulse of ENU (50 mg/kg B. W.) to pregnant SD-JCL rats on the 18th day of gestation. Primary cultured cells were divided into 20 groups, and graded doses of PB from 1.0 mM to 5.0 mM or that of TPA from 0.1 ng/ml to 50.0 ng/ml were added continuously into cultured medium. In the ENU group treated with TPA, it was found the results of more early appearance of "piled up focus" for about 90 days, more early capacity of colony formation in semisolid soft agar for about 110 days, and more early appearance of tumorigenicity for about 90 days than that in the ENU groups treated with or without acetone. An increased agglutinability to Concanavalin A (200 micrograms/ml) was appeared more early in the ENU groups treated with TPA than in the ENU groups. On the basis of these results, it is suggested that TPA promoted the in vitro malignant transformation of the fetal rat brain cells with a transplacental carcinogen of ENU, and TPA might effect as a tumor promoter on the neurogenic carcinogenesis. As for the mechanisms of the promoting effect of TPA, it is speculated that TPA might modulate the function of cell membrane from the results of Concanavalin A agglutinability, and might modulate the differentiation on cell function from the results of morphological analysis.