Hydroxyproline (HP) can be quantified by the sensitive colorimetric procedure of Kivirikko et al. (1967). Without preliminary hydrolysis, only the free imino acid (I) can be detected. After hydrolysis in 6 N HCl at 120 C for 15 hr, also the peptide‐bound (II) and polypeptide‐bound (III) is detected. Cells grown in 2% (w/v) proteose peptone supplemented with 0.4% yeast (w/v) extract (PPY) contained 0.01 mg HP/mg cellular proteins. Over 95% was in the I or II form (soluble in cold 20% trichloro acetic acid). Cells grown in a chemically defined medium (DM), contained less than 0.34 μg/mg cellular proteins. Whereas the DM does not contain any HP, the PPY medium is rich in HP (0.032 mg/mg proteose peptone). In conclusion, the HP found in the cells grown on PPY is a “contaminant'’from this medium. No endogenous production of HP was demonstrated.