We have used a high density microarray hybridization approach to characterize the transcriptional response of human hepatoma HepG2 cells to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We find that exposure to 10 nM TCDD for 8 hr alters by at least a factor of 2.1 the expression of 310 known genes and of an equivalent number of expressed sequence tags. Treatment with TCDD in the presence of 20 microg/mL of cycloheximide blocked the effect on 202 of these genes, allowing us to distinguish between primary effects of TCDD exposure, which take place whether cycloheximide is present or not, and secondary effects, which are blocked by inhibition of protein synthesis. Of the 310 known genes affected by TCDD, 30 are up-regulated and 78 are down-regulated regardless of cycloheximide treatment, and 84 are up-regulated and 118 are down-regulated only when protein synthesis is not inhibited. Functional clustering of genes regulated by TCDD reveals many potential physiological interactions that might shed light on the multiple biological effects of this compound. Our results, however, suggest that arriving at a sound understanding of the molecular mechanisms governing the biological outcome of TCDD exposure promises to be orders of magnitude more complicated than might have been previously imagined.