Indole-3-carbinol (I3C), a compound that occurs naturally in Brassica vegetables such as cabbage and broccoli, can induce a G1 cell-cycle arrest of human MCF-7 breast cancer cells that is accompanied by the selective inhibition of cyclin-dependent kinase 6 (Cdk6) expression and stimulation of p21(Waf1/Cip1) gene expression. Construction and transfection of a series of promoter-reporter plasmids demonstrate that the indole-regulated changes in Cdk6 and p21(Waf1/Cip1) levels are due to specific effects on their corresponding promoters. Mutagenic analysis reveals that I3C signaling targets a composite transcriptional element in the Cdk6 promoter that requires both Sp1 and Ets transcription factors for transactivation function. Analysis of protein-DNA complexes formed with nuclear proteins isolated from I3C-treated and -untreated cells demonstrates that the Sp1 DNA element in the Cdk6 promoter interacts with an I3C-inhibited protein-protein complex that contains the Sp1 transcription factor. In indole-treated cells, a fraction of [(3)H]I3C was converted into its natural diindole product (3)H-labeled 3-3'-diindolylmethane ([(3)H]DIM), which accumulates in the nucleus; this suggests that DIM may have a role in the transcriptional activities of I3C. Mutagenic analysis of the p21(Waf1/Cip1) promoter reveals that in transfected breast cancer cells, DIM (as well as I3C) stimulates p21(Waf1/Cip1) transcription through an indole-responsive region of the promoter that contains multiple Sp1 consensus sequences. Furthermore, DIM treatment regulates the presence of a nuclear Sp1 DNA-binding activity. Our results demonstrate that both the Cdk6 and p21(Waf1/Cip1) promoters are newly defined downstream targets of the indole-signaling pathway, and that the observed transcriptional effects are due to a combination of the cellular activities of I3C and DIM.