DNA adducts produced in vivo in rat lung, liver, and peripheral blood lymphocytes following the i.p. administration of several synthetic benzo(a)pyrene (B(a)P) metabolites and ring-substituted derivatives have been analyzed by the nuclease P1 version of the 32P-postlabeling assay. These include 1-,2-,3-,4-,5-,6-,7-,8-,9-,10-,11-, and 12-hydroxyB(a)P, (+ or -) -B(a)P-trans-4,5-dihydrodiol, (+ or -) -B(a)P-trans-7,8-dihydrodiol, (+ or -) -B(a)P-trans-9,10-dihydrodoil, and B(a)P-7,8-dione. Among the monohydroxy derivatives, only 2-,9-, and 12-hydroxyB(a)P produced detectable adducts. The only disubstituted derivative studied which produced adducts was the trans-7,8-dihydrodiol. The resulting DNA adducts were compared to those produced in each tissue by administration of B(a)P. 9-hydroxyB(a)P and B(a)P-trans-7,8-dihydrodiol each lead to the formation of major B(a)P adducts seen in lung and liver, respectively. None of the adducts derived from either 2-hydroxyB(a)P or 12-hydroxyB(a)P were observed following administration of B(a)P alone.