The placental membrane radioreceptor assay was used to measure the levels of somatomedin (SM) peptides in plasma. Displacement of both [125I]somatomedin A ([125I]SM-A) and [125I]somatomedin C ([125I]SM-C) by normal whole plasma, the peptide fraction of acid-chromatographed plasma, and a partially purified, insulin-free SM preparation were compared. The peptide fraction of plasma was isolated by acid chromatography over Sephadex G-50 in 0.25 M formic acid with a yield of greater than or equal to 90%, as determined by bioassay and [125I]SM. In the case of [125I]SM-A, the dose-response curves for whole plasma, acid-chromatographed plasma, and the standard SM preparation were parallel (P less than 0.2). In contrast, for [125I]SM-C, the dose-response curves for acid-chromatographed plasma and the purified SM preparation were parallel (P less than 0.2), but both differed significantly from that of whole plasma (P less than 0.001). In addition, there was less variability in the assay of acid-chromatographed plasma compared to whole plasma. The results indicate that radioreceptor assay of unextracted normal plasma using [125I]SM-A is a valid measure of SM peptide concentration, while radioreceptor assay of unextracted normal plasma using [125I]SM-C, in our hands, is not. Acid chromatography of plasma before its assay is an uncomplicated procedure which allows valid and precise measurement of SM peptide content using either [125I]SM-A or [125I]SM-C.