Dithiolethiones inhibit tumorigenicity elicited by many structurally diverse carcinogens in numerous target tissues. These protective actions are associated with the induction of several carcinogen detoxification enzymes, some of which have only recently been discovered. In order to identify additional novel inducible detoxification response genes, a cDNA library was prepared from liver of rats treated with 1,2-dithiole-3-thione (D3T) and was screened by a differential hybridization method. Complementary DNA clones for several known D3T-inducible genes were isolated, such as epoxide hydrolase, aflatoxin B1-aldehyde reductase, quinone reductase and multiple subunits of glutathione S-transferase. Clones representing genes not previously associated with detoxification were isolated, including those for ferritin heavy and light subunits, ribosomal proteins L18a and S16 and two novel genes, termed dithiolethione-inducible genes (or DIG-1 and DIG-2). Levels of mRNA recognized by each clone were increased from 2- to 31-fold, with maximum induction between 6 and 30 h after treatment with D3T. Except for epoxide hydrolase, the kinetics of induction of each mRNA was coordinate with increased rates of gene transcription. However, based on the time of response to D3T, at least two sets of responsive genes were identified. One set of genes, including glutathione S-transferase Yp, aflatoxin B1-aldehyde reductase, quinone reductase and DIG-1, had low constitutive and highly inducible expression (approximately 20-fold) and the other, including glutathione S-transferase Ya and Yb, epoxide hydrolase, ferritin heavy and light subunits, ribosomal proteins L18a and S16 and DIG-2, had relatively high constitutive and modestly inducible expression (approximately 5-fold). The simplest explanation for this differential expression of D3T-inducible genes is that multiple regulatory mechanisms govern their response. The transcriptional activation of ferritin, ribosomal protein, DIG-1 and DIG-2 genes in conjunction with those of carcinogen detoxification enzymes suggests that they participate in the pleiotropic cellular defense response to dithiolethiones that inhibits chemically produced tumorigenesis.