The metabolism of hydroxyproline by the rat kidney leads to the production of significant quantities of both glycine and serine. This process was observed in both the isolated perfused kidney and in isolated cortical tubule suspensions. The rate of hydroxyproline metabolism was increased in both preparations by the addition of alanine. The distribution of hydroxyproline oxidase, hydroxyoxoglutarate aldolase and alanine-glyoxalate transaminase were determined in detail. All three enzymes were found exclusively in the renal cortex where they were restricted to the mitochondria. Cortical tubule fractionation studies indicated that the enzymes are located in the proximal convoluted and proximal straight segments at the nephron. The results suggest that hydroxyproline degradation could contribute significantly to the renal synthesis of serine.