The mechanism responsible for the osmotic sensitivity of sarcosine oxidation by liver mitochondria from the little skate, Raja erinacea Mitchill, is examined. Assay medium tonicity, rather than a solute effect (urea or trimethylamine oxide), is probably responsible for the inverse relationship between osmolarity and the rate of oxidation of sarcosine by these mitochondria. Sarcosine oxidation proceeds through the flavin-linked sarcosine oxidase with the resultant glycine catabolized in the NAD-linked glycine cleavage system. The tonicity-sensitive component of the sarcosine oxidative pathway is not the glycine cleavage system. Sarcosine oxidation in the presence of rotenone is sensitive to medium tonicity. Oxidation of serine, which is also catabolized through the glycine cleavage system, is not as sensitive to tonicity as is sarcosine oxidation. Mitochondrial volume changes also appear to affect the transport of glycine. Although sarcosine does not appear to share the glycine transporter, it is possible that sarcosine transport is similarly sensitive to medium tonicity. The effects of osmolarity on the oxidation of dimethylglycine appear to support this hypothesis. Tonicity effects on sarcosine oxidase cannot yet be eliminated.