Peptides derived from the aminoterminal portion of the palpha1(I)-chain of calf and sheep procollagen were labeled with iodine-125. Despite changes in electrophoretic homogeneity after labeling, reaction of the labeled peptide with antisera to unlabeled peptide was retained. Antisera to procollagen or the isolated procollagen peptide showed high titers for the native peptide, a much weaker binding with the reduced and alkylated peptide and little or no reaction with collagen. Antisera to collagen showed strong binding with collagen and a weaker but distinct reaction with the procollagen peptide. Evidence was obtained that a minor contaminant of procollagen peptide was present in the acid-extracted collagen and that there were no shared antigenic determinants. Bovine serum and amniotic fluid contained 1-10 mug/ml reactive antigen. These results indicate that the labeled peptides can be used as a specific, sensitive and accurate assay for the amino-terminal portion of procollagen in biological samples.