Consensus sequences at the splice donor, splice acceptor, and lariat branch point regions are necessary but insufficient determinants of splice-site selection in nuclear precursor mRNAs. Sequences outside of these regions can have a significant effect on the utilization of splice sites. Although the mode of action of such sequences is undefined in most cases, higher order RNA structures have been suggested as a potential contributor to splice-site selection. During a detailed analysis of the splicing patterns of the human growth hormone transcript, we located 2 bases in the vicinity of the exon 3 major splice-acceptor site (B) which facilitate the utilization of a competing downstream acceptor (B'). The effects of a series of site-specific mutations on the splicing pattern demonstrate that these 2 bases function by stabilizing a specific stem-loop structure in the native transcript. This defined secondary structure selectively encompasses the upstream B splice-acceptor site together with its lariat branch point region. Increasing the predicted stability of this stem by point mutations results in a corresponding shift in splicing towards the alternative B' splice-acceptor site. These results indicate that a specific secondary structure within the native human growth hormone transcript controls the relative utilization of two competing splice-acceptor sites with the consequent generation of two functionally distinct hormone isoforms.