The tritiated chiral alkanes (S)-[1-H-2(1),1-H-3]ethane, (R)-[1-H-2(1),1-H-3]ethane, (S)-[1-H-2(1),1-H-3]butane, (R)-[1-H-2(1),1-H-3]butane, (S)-[2-H-3]butane, (R)-[2-H-3]butane, and racemic [2-H-3]butane were oxidized by soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath), and the absolute stereochemistry of the resulting product alcohols was determined in order to probe the mechanism of substrate hydroxylation. When purified hydroxylase, coupling protein, and reductase components were used, the product alcohol displayed 72% retention of stereochemistry at the labeled carbon for the ethane substrates and 77% retention for the butanes labeled at the primary carbon. A putative alkyl radical which would yield these product distributions would have a lifetime of 100 fs, a value too short to correspond to a discrete intermediate. Intramolecular k(H)/k(D) ratios of 3.4 and 2.2 were determined for ethane and butane, respectively. When the hydroxylations were performed with purified hydroxylase but only a partially purified cellular extract for the coupling and reductase proteins, different product distributions were observed. These apparently anomalous results could be explained by invoking exchange of hydrogen atoms at the ct carbon of the product alcohols. The characteristics of this exchange reaction are discussed. Hydroxylation of [2-H-3]butanes by the latter system yielded similar to 90% retention of stereochemistry at the labeled carbon. The implication of these results for the catalytic mechanism of sMMO is discussed. Together with the mechanistic information available from a range of substrate probes, the results are best accounted for by a nonsynchronous concerted process involving attack on the C-H bond by one or more of several pathways discussed in the text.