Loss of tritium from specific positions in [3H,14C] aromatic hydrocarbons can elucidate their binding site(s) to DNA and RNA and indicate the mechanism of activation. Studies of tritium loss from [6-3H,14C]benzo[a]pyrene (B[a]P), [1,3-3H,14C]B[a]P, [1,3,6-3H,14C]B[a]P, [6,7-3H,14C]B[a]P, and [7-3H,14C]B[a]P were conducted in vitro using liver nuclei and microsomes from 3-methylcholanthrene-induced Sprague-Dawley rats and in vivo on the skin of Charles River CD-1 mice. The relative loss of tritium from [3H, 14C]B[a]P was measured after binding to skin DNA and RNA, to nuclear DNA, and to native and denatured calf thymus and rat liver DNA's and poly(G) by microsomal activation. In skin, nuclei, and microsomes plus native DNA, virtually all B[a]P binding occurred at positions 1,3 and 6; while with microsomes plus denatured DNA or poly(G), B[a]P showed no binding at the 6 position and a small amount at the 1 and 3 positions. In vivo and with nuclei, binding at the 6 position predominated. Little loss of tritium from the 7 position was seen; this was expected because binding at this position is not thought to occur. This confirms the interpretation of loss of tritium as an indication of binding at a given position. These results demonstrate that the use of microsomes to activate B[a]P is not a valid model system for delineating the in vivo mechanism of B[a]P activation, and support previous evidence for one-electron oxidation as the mechanism of activation of hydrocarbons in binding to nucleic acids.