Multiphoton laser scanning microscopy (MPLSM) is a promising tool to study the tissue distribution of environmental chemical contaminants during fish early life stages. One such chemical for which this is possible is benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon that absorbs strongly at UV wavelengths and fluoresces following multiphoton excitation. BaP is enzymatically converted to hydroxylated metabolites, which are further modified to more polar conjugates. To determine whether fluorsecent signal from parent compound and metabolites could be differentiated by MPLSM, multiphoton excitation spectra were determined from 730-880 nm using a tunable Ti:Sapphire laser. BaP-3-hydroxy (BaP-3-OH) was the most fluorescent and the two conjugated metabolites, BaP-3-sulfate and BaP-3-glucuronide, exhibited fluorescence intensity intermediate between BaP and BaP-3-OH. For example, at 760 nm the fluorescence of conjugated metabolites was four-fold greater, while BaP-3-OH exhibited 16-fold greater fluorescent intensity than BaP. At wavelengths longer than 830 nm there was no excitation of BaP above background. Spectral differences at the longer wavelengths were used to detect the presence of the primary metabolite BaP-3-OH in the presence of parent or conjugated metabolites in fish egg homogenates. Thus, multiphoton excitation spectral characteristics can provide a means to follow the tissue distribution of parent and metabolite in developing fish.