A simple and practical method for the quantitation of nanogram levels of 4-hydroxyproline in tissue hydrolysates is described. Optimum conditions were employed to release the hydroxyproline from tissue homogenates by acid hydrolysis. The free hydroxyproline was then oxidized by chloramine T to produce a pyrrole-type compound. The addition of Ehrlich's reagent resulted in the formation of a chromophore with a wavelength maximum at 558 nm. The optimum conditions for the assay, notably the chloramine T concentration, were different for pyrrole and hydroxyproline standards, and tissue hydrolysates. It is shown that the calculation of recoveries based on the external standards method using either pyrrole or hydroxyproline could be subject to considerable error. This necessitated the use of the method of standard additions. The recovery of hydroxyproline under these conditions was 97 ± 8%. No substantial matrix effect exists with hydrolysate quantities ranging from 25 to 100 μl corresponding to 1.2 to 5 mg of tissue (wet wt). The hydroxyproline content of adult rat lung was consistently found to be 3.78 ± 0.15 μmol/g wet wt.