Translational regulation of the protamine 1 mRNA is mediated by sequences in its 3' untranslated region. In this study, we demonstrate that a highly conserved sequence, the translational control element, is solely responsible for protamine 1 translational regulation. Mutation of the conserved sequence causes premature translation of a transgene containing a fusion between the human growth hormone coding sequence and the protamine 1 3' untranslated region. Temporal expression of the transgene was monitored in prepubertal animals by Northern and Western blotting and in adult animals by immunocytochemistry. Messenger RNAs lacking the translational control element sediment in the messenger ribonucleoprotein particle and ribosomal fractions of polysome gradients, suggesting that the translational control element is required for translational repression but not for incorporation of mRNAs into ribonucleoprotein particles.