We have investigated several methods for determining rates of xenobiotic biotransformation in individual rabbit hepatocytes by microspectrofluorometry. Experiments designed to measure monooxygenase activity by following fluorescent product formation (i.e. 7-ethoxycoumarin deethylase or oxidation of the fluorescein derivatives ethoxyfluorescein ethyl ester and 5- and 6-ethoxycarbonyl ethoxyfluorescein ethyl ester) demonstrated that the fluorescent products were released from cells to the surrounding media. Thus, metabolically active cells probably had underestimated activities, and inactive cells could accumulate fluorescent product and appear metabolically active. We also utilized benzo(a)pyrene (BP) as a substrate in our system. By selecting appropriate wavelengths (370 nm excitation, 407 nm emission), it was possible to selectively monitor BP disappearance in single cells. In hepatocytes from rabbits, disappearance of fluorescence was observed after a 10-min incubation at 37 degrees C with 1-40 microM BP followed by a wash to remove extracellular substrate. The decrease in fluorescence followed apparent first-order kinetics. Measurement of extracellular fluorescence indicated that the substrate did not leave the hepatocytes. Furthermore, in both control and beta-naphthoflavone-treated rabbits, the observed fluorescence changes in the cells were inhibited by the presence of equimolar concentrations of ellipticine or alpha-naphthoflavone, inhibitors of BP metabolism. Storage of hepatocytes from untreated animals on ice for 3.5 hr did not affect rate constant values in cells with a normal appearance, whereas those lacking refringent edges or having numerous cytoplasmic blebs demonstrated greatly reduced (up to 88% loss) activities compared with normal, freshly isolated cells. Potential applications of this system (e.g. to other cell types) are discussed.