Total RNA extracted from a rat liver cell line (BRL 3A) that synthesizes rat insulin-like growth factor II (IGF-II) was translated in a reticulocyte lysate cell-free system. Incubation of the translation products with antisera to mature rat IGF-II (Mr 8700) selectively immunoprecipitated a Mr 21,600 protein. We propose that this protein represents pre-pro-IGF-II.