Abstract When matrix-free tendon cells from chick embryos were incubated with [14C]proline and then extracted with sodium dodecyl sulfate and mercaptoethanol, a major part of the newly synthesized 14C-protein was found to consist of collagen polypeptides which were complete in that their content of [14C]hydroxyproline and their size was the same as that of polypeptides of the precursor form of collagen secreted into the medium. When the cells were incubated with 0.3 mm α,α'-dipyridyl so as to inhibit protocollagen proline hydroxylase and protocollagen lysine hydroxylase, secretion of 14C-protein was inhibited and [14C]protocollagen accumulated within the cells. [14C]Protocollagen extracted from the cells was comprised of polypeptides which were of the same size as polypeptides of the intracellular collagen or about 125,000 as estimated by gel filtration in sodium dodecyl sulfate. In contrast, the small amount of peptide-bound 14C still secreted in the presence of α,α'-dipyridyl was shown to consist of small peptides which were in part derived from the intracellular degradation of [14C]protocollagen. The results demonstrated therefore that in freshly isolated tendon cells protocollagen itself is not secreted. Since the rate of protein synthesis in the presence of α,α'-dipyridyl was the same as under control conditions for about 90 min, the data suggested that the intracellular [14C]protocollagen accumulated in some postribosomal compartment. Extraction of control cells with acetic acid solubilized a large fraction of the intracellular collagen, and amino acid analyses indicated that 40 to 50% of the protein in the extracts was collagen. The protein extracted from cells incubated with α,α'-dipyridyl was similar in amino acid composition, but it contained essentially no hydroxylysine and hydroxyproline and was correspondingly rich in lysine and proline. In addition, 40% of the proline in the protein extract was converted to hydroxyproline after incubation with pure protocollagen proline hydroxylase. Experiments involving limited pepsin digestion provided the first demonstration that intracellular collagen and proto-collagen are largely in a native, triple-helical conformation. After acetic acid extracts from control cells and from cells incubated with α,α'-dipyridyl were dialyzed against ATP, segment long spacing aggregates were obtained. The aggregates were similar to those formed by extracellular, fibrillar collagen except that they had a 130-A NH2-terminal extension which was indistinguishable from that seen in aggregates of the precursor form of collagen secreted by the same cells.