Second trimester human amniotic fluid cells synthesize and secrete a variety of collagenous proteins in culture. F cells (amniotic fluid fibroblasts) are the most active biosynthetically and synthesize predominantly type I with smaller amounts of type III procollagen. Epithelioid AF cells (the predominating clonable cell type) synthesize a type IV-like procollagen and a procollagen with three identical proa chains, structurally and immunologically related to the proαl chains of type I procollagen. The latter procollagen, when cleaved with pepsin and denatured, yields a single non-disulfide-bonded a chain that migrates more slowly than F cell or human skin α1 (I) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis but coelutes with these chains from carboxymethylcellulose. The major cyanogen bromide produced peptides demonstrate a similar behavior relative to peptides derived from αl(I). The collagen is characterized by an increased solubility at neutral pH and high ionic strength, relative to type I collagen. The amino acid composition of the pepsin-resistant a chain is essentially identical with that of human αl (I), except for marked increases in the content of 3- and 4-hydroxyproline and hydroxylysine. Preliminary experiments suggest that these increased posttranslational modifications are responsible for the unusually slow migration of this collagen and its cyanogen bromide peptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The procollagen has, therefore, been assigned the chain composition [proαl(I)]3. Like type I procollagen, [proαl(I)]3 undergoes a time-dependent conversion, in the medium and cell layer, to procollagen intermediates and α chains. The production of [proαl(I)]3 probably reflects the state of differentiation and/or embryologic derivation of AF cells rather than a characteristic of the fetal phenotype, since F cells do not synthesize significant amounts of the procollagen.