The two‐component alkanesulfonate monooxygenase systems, consisting of a flavin reductase (SsuE and MsuE) and an alkanesulfonate monooxygenase (SsuD and MsuD), enable bacterial organisms to utilize a broad range of alkanesulfonates when sulfur is limiting. The flavin reductases supply reduced flavin to their partner monooxygenase for the desulfonation of sulfonated compounds through the formation of a flavin oxygenating intermediate. Commonly, flavin monooxygenases have been proposed to utilize a C4a‐(hydro)peroxyflavin as the oxygenating intermediate. However, unlike other flavin monooxygenase enzymes, this intermediate has not been spectrally observed in the alkanesulfonate monooxygenase enzymes. Recent studies reported the formation of a flavin‐N5‐oxide which forms as an intermediate during turnover or as the final product in some flavin monooxygenases. It is hypothesized that the alkanesulfonate monooxygenases could employ a flavin‐N5‐adduct based on amino acid sequence alignments and structural similarities with enzymes employing similar intermediates.SsuD and MsuD share comparable structural properties but have different specificities for alkanesulfonate substrates. Although the substrate preference is critical for catalytic competence, SsuD and MsuD likely utilize similar catalytic steps for desulfonation. Previous studies identified various polar and nonpolar amino acid residues that were critical for the formation and stabilization of the flavin‐N5‐oxide. The conserved nonpolar amino acids are proposed to control the interaction of the flavin‐N5 with molecular oxygen, whereas the polar amino acids are involved in stabilizing the superoxide anion involved in the formation of the flavin‐N5 oxygenating intermediate. Some of these amino acids were also found to be conserved in the alkanesulfonate monooxygenases and their role was further evaluated through site‐directed mutagenesis. The V108T and T109A SsuD variants had similar activity to wild‐type SsuD; however, the N108L SsuD variant had no measurable activity. These results support the role of Asn in stabilizing the proposed flavin‐N5 oxygenating intermediate. Since a flavin‐N5‐oxide has been identified as an intermediate or as the final product in some two‐component monooxygenases, HPLC and mass spectrometric analyses were performed to determine if the alkanesulfonate monooxygenases form the flavin‐N5‐oxide. To provide evidence for kinetic steps and identify flavin‐N5 reaction intermediates, stopped‐flow kinetic experiments were performed with wild‐type and variants of SsuD and MsuD. The findings from this study provide insight into the mechanistic features of the alkanesulfonate monooxygenases as well as their role in the overall global sulfur cycle.