Nitrosopyrrolidine (NO-PYR), an hepatocellular carcinogen, is rapidly metabolized to CO2 by hepatocytes freshly isolated from the livers of male Fischer rats. Using CO2 evolution as a measure of NO-PYR metabolism, we observed two kinetic constants; a high affinity component (Km = 0.11 mM), and a lower affinity component (K m = 3.2 mM). The high affinity component has similar kinetic constants to those observed for in vitro reactions with microsomes plus cytosol (Km = 0.36 mM). Therefore, it is probable that the microsomal reaction is the limiting factor in the metabolism of NO-PYR in hepatocytes. NO-PYR may be metabolized to CO2 through normal anaplerotic sequences. Some metabolites of NO-PYR which have been tentatively identified are gamma-hydroxybutyrate, succinic semialdehyde, 3,4-dihydroxybutyric acid lactone, lactate, acetate, pyruvate, glyoxylate, gamma-aminobutyrate and alanine. 2-Hydroxytetrahydrofuran (2-hydroxy-THF). a product of alpha-hydroxylation was detected at low levels in only one of four reactions. 3-Hydroxy-NO-PYR is present but represents only a small percentage of the total metabolism and is probably of little significance in the overall catabolism of NO-PYR in hepatocytes.