Quantification of polycyclic aromatic hydrocarbons (PAH) and their metabolites within living cells and tissues in real time using fluorescence methods is complicated due to overlaping excitation and/or emission spectra of metabolites. In this study, simultaneous analysis of several metabolites of a prototype carcinogenic PAH, benzo[a]pyrene (BaP) in undifferentiated (MCF10A) and differentiated (MCF10CA1h) breast cancer cells was performed using single-cell multiphoton spectral analysis. The two cell types were selected for this study because they are known to have differences in BaP uptake and metabolism and induction of aryl hydrocarbon receptor-dependent ethoxyresorufin-O-deethylase (EROD) activity. Multiphoton microscopy spectral analysis performed in cells exposed to BaP for 24 hr identified 5 major peaks of fluorescence that were monitored within spectral bands. A comparison of the fluorescence peaks within these bands to those of BaP metabolite standards indicated that a peak in the spectral range of 393-415 nm matched benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide(+/-),(anti) (BPDE), the ultimate carcinogenic BaP metabolite. In addition, the 426-447 nm band matched the major metabolites 3-hydroxybenzo[a]pyrene (3-OH BaP) and 9-hydroxybenzo[a]pyrene (9-OH BaP); the 458-479 nm band corresponded to the secondary metabolite benzo[a]pyrene-3,6-dione (3,6 BPQ); and a peak at 490-530 nm matched the parent compound, BaP. Multiphoton spectral analysis also revealed differences in fluorescence intensities between MCF10A and MCF10CA1h cells within three spectral bands: 393-415 nm, 426-447 nm and 458-479 nm which were partially reversed with cyclosporine A suggesting differences in efflux mechanisms between cell lines. These results demonstrate the feasibility of analyzing BaP metabolism in situ by multiphoton spectral analysis and also identifying cell-type differences in BaP accumulation and metabolism.