mRNA secondary structure can be an important determinant of the efficiency of translation initiation. To study the effect of secondary structure on translation initiation, in vitro secondary structure analysis was performed on 32 lacZ RNA transcripts that differ in their in vivo translation initiation efficiencies because of mutations. We have shown that well-translated RNA has a relatively unstructured translation initiation region in vitro. In contrast, the translation initiation region of many of the poorly translated RNA transcripts is involved in a stem-loop structure. Mutations that decrease the in vitro stability of the stem-loop increase the frequency of translation initiation. The sequences responsible for forming this stem-loop structure were localized to a small region of RNA. The results confirm some of the previous predictions of the RNA secondary structure of the mutant RNAs based on computer modeling, but they disagree with some of the predicted long-range interactions.