Human type II procollagen was prepared in a recombinant system and cleaved to pC-collagen II by procollagen N-proteinase. The pC-collagen II was then used as a substrate to generate collagen II fibrils by cleavage with procollagen C-proteinase at 37 degrees C. Electron microscopy of the fibrils demonstrated that, at the early stages of fibril assembly, very thin fibrils were formed. As the system approached equilibrium over 7-12 h, however, the thin fibrils were largely but not completely replaced by thick fibrils that had diameters of about 240 nm and a distinct D-period banding pattern. One typical fibril was photographed and analyzed in its entirety. The fibril was 776 D-periods (52 microM) long. It had a central shaft with a uniform diameter that was about 516 D-periods long and two tips of about 100 D-periods each. Most of the central shaft had a symmetrical banding pattern flanked by two transition regions of about 30 D-periods each. Measurements by scanning transmission electron microscopy demonstrated that the mass per unit length from the tips to the shafts increased linearly over approximately 100 D-periods from the fibril end. The linear increase in mass per unit length was consistent with previous observations for collagen I fibrils and established that the tips of collagen II also had a near paraboloidal shape. However, the orientation of monomers in the tips differed from the tips of collagen I fibrils in that the C termini instead of the N termini were directed toward the tips. The thin fibrils that were present at early stages of assembly and at equilibrium were comparable to the collagen II fibrils seen in embryonic tissues and probably represented intermediates on the pathway of thick fibrils formation. The results indicated that the molecular events in the self-assembly of collagen II fibrils are apparently similar to those in self-assembly of collagen I fibrils, but that there are also important differences in the structural information contained in collagen I and collagen II monomers.