We have previously shown that methotrexate (MTX) and hydroxyurea (HU) stimulate expression of the human chorionic gonadotropin alpha and placental alkaline phosphatase genes and repress the expression of the c-myc oncogene in BeWo choriocarcinoma cells. In order to determine whether c-myc downregulation played a role in the induction of these placental genes, we treated BeWo choriocarcinoma cells with aphidicolin (APH), and 6-diazo-5-oxo-L-norleucine (DON), and compared the effects of these drugs with that of MTX. All of these drugs downregulate c-myc gene expression. At the doses used, both APH and DON repress c-myc expression to approximately the same extent, as well as inducing morphologic changes in BeWo similar to that of MTX, although neither stimulated hCG alpha expression. Both DON and APH stimulate placental alkaline phosphatase gene expression, but only MTX and DON stimulate cholesterol side chain cleavage enzyme gene expression. This indicates that the downregulation of c-myc gene expression is insufficient to stimulate the expression of all the placental genes stimulated by MTX.