Human placentas express high levels of the placental alkaline phosphatase (PLAP) gene and low levels of a highly related gene, germ cell AP (GCAP). Malignant transformation of the placenta is accompanied by a reversal of this pattern of expression. Three Sp1-binding GC-rich DNA elements (sites I-III) located within the first 156 base pairs upstream of the GCAP gene have been shown to direct optimal GCAP gene expression in choriocarcinoma cells. Here we show that the first 100 base pairs upstream of the GCAP gene, which contains sites I and II, constitutes a minimal GCAP promoter. The simultaneous presence of both sites I and II is necessary for GCAP expression and its induction by sodium butyrate. The PLAP promoter directs only a very low level of gene expression in choriocarcinoma cells; the expression does not respond to butyrate. The -100/-1 DNA regions between the GCAP and PLAP promoters differ by only eight base pairs. However, the GC-rich stretches in sites I and II of the GCAP promoter are disrupted in the corresponding PLAP promoter. This disruption blocks or markedly reduces the binding of choriocarcinoma nuclear factors to the PLAP promoter, leading to a reduction in expression and a loss of butyrate response. We further demonstrate that nucleotides -75 to -58 in both AP promoters, which bind a human Y-box binding protein, appear to down-regulate GCAP expression.