A study was performed to identify the first translated product of messenger RNA for retinol-binding protein (RBP), the specific plasma transport protein for vitamin A. Poly(A)+RNA was isolated from rat liver and translated in the rabbit reticulocyte in vitro protein-synthesizing system. RBP was identified and separated from other translated products by immunoprecipitation with specific rabbit anti-rat RBP antiserum. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of the immunoprecipitate consistently revealed one major product which migrated more slowly than purified rat serum RBP. The protein (preRBP) had an approximate molecular weight of 24,000. When dog pancreas microsomal membranes were cotranslationally present, the newly synthesized preRBP was processed to a protein which migrated coincidentally with purified rat serum RBP, approximately 20,500 daltons. These results indicate that RBP is initially synthesized as a larger molecular weight precursor (preRBP) which is rapidly processed by the removal of a peptide of approximately 3,500 daltons to the size of the final RBP molecule that circulates in the plasma.