The human teratocarcinoma cell NTERA-2 cl. D1 (NT2/D1) is a cloned embryonal cancer cell line that differentiates into a neuronal phenotype and other cellular lineages after treatment with retinoic acid (RA). We examined the regulated expression of growth factors and proto-oncogenes in NT2/D1 cells. We studied RNA levels after six days of RA treatment to assess gene expression coincident with observed morphologic differentiation. Three growth factors were markedly down-regulated following RA treatment: Hst-1/kFGF and TGF-alpha expression became undetectable by Northern analysis and bFGF expression was substantially reduced. Minimal decline was seen for c-myc, N-myc, c-fos, and c-myb. Increased expression with differentiation was seen for the human homeotic genes Hox 2.1 and Hox 2.2. Assay of RNA levels daily after one to six days of RA treatment showed that the growth factor down-regulation inversely correlated with the homeotic gene up-regulation. Nuclear run-on studies showed low transcriptional rates for these homeotic genes, Hst-1/kFGF, and TGF-alpha that did not measurably change with RA treatment. To explore whether these regulated genes in NT2/D1 play a role in human testicular cancer, we examined RNA levels in a panel of human germ cell cancer lines. Hst-1/kFGF and bFGF are commonly expressed in five of seven male germ cell cancer lines. These data show that specific proto-oncogenes and growth factors are down-regulated with RA treatment of the NT2/D1 cell and that some of these regulated genes are often expressed in human germ cell cancer lines.