The effect of a methyl substituent at the non-benzo ring bay-region position of benzo[e]pyrene (B[e]P), cholanthrene (CA) and dibenz[a,j]anthracene (DB[a,j]A) on skin tumor-initiating activity was examined. A methyl substituent at the 1-carbon of B[e]P enhanced tumor-initiating activity of the parent compound (0.18 vs. 2.4 papillomas/mouse for B[a]P vs. 1-methyl-B[e]P, respectively, with an 800 nmol initiating dose). A methyl substituent at the 7- and 14-carbons of DB[a,j]A increased the activity of this weak initiator more than 13 times. The introduction of a methyl substituent at the non-benzo ring bay-region position of CA (i.e. carbon atom 6) dramatically increased tumor-initiating activity in SENCAR mice. 6-Methyl-CA was found to be a more potent skin tumor-initiator than 3-methyl-CA, and nearly as potent as 7,12-dimethylbenz[a]anthracene, one of the most potent initiators yet tested in the 2-stage initiation-promotion mouse skin model. When taken together with previous results from our laboratories, the data further support the generality of the enhancing effect of a non-benzo ring bay-region methyl substituent on polycyclic hydrocarbon tumor-initiation.