Hydroxylysyl pyridinoline (HP) is a nonreducible collagen crosslink derived from three posttranslationally modified lysyl residues. Neonatal rat aorta smooth muscle cell cultures (NNRSMC) produce mg amounts of insoluble collagen. The purpose of the present study was to evaluate the capability of NNRSMC to produce collagen containing HP crosslinks. Cultures were pulsed with [14C]-lysine and then chased for five weeks. Insoluble collagen was prepared by digestion of the cell layer material with porcine pancreatic elastase and trypsin. After acid hydrolysis and cation-exchange chromatography, purified HP was isolated by reversed phase ion-paired chromatography. The material eluting from the HPLC was monitored continuously at 295 nm and the ultraviolet absorption spectrum was recorded every 21 msec. The ultraviolet spectrum of the HP peak was virtually identical to that of standard HP run on the HPLC. The HP exhibited a homogeneity of 97.3% when the ultraviolet spectrum of the apex of the peak was compared with the spectra of the shoulders of the peak. The radioactive HP also exhibited the expected fluorescence emission spectrum. We calculate a mean of 0.40 +/- 0.03 nmol HP/nmol collagen in the three experiments as compared with reported values of 0.57 +/- 0.1 for rabbit aorta. This is the first report of cell culture biosynthesis of chemically measurable amounts of HP. Using such pulse-chase techniques one can study the maturation of intermediate collagen crosslinks into HP. HP can also be used as a marker to study the metabolism of mature collagen molecules during normal and pathologic states.