The large ATP synthase gene cluster from spinach (Spinacia oleracea) chloroplasts encodes five genes, the last four of which encode subunits of the ATP synthase complex. In preliminary experiments (J.K. Kim, M.J. Hollingsworth [1992] Anal Biochem 206: 183-188) it was shown that ribosomes pause during translation of these open reading frames. We have examined ribosome pausing in the four ATP synthase open reading frames of this gene cluster to determine whether it could affect the final ratio of the ATP synthase polypeptides derived from the cluster. Ribosome pauses were mapped and found to be distributed in a nonuniform manner. We have quantitated the relative extent of ribosome pausing within each open reading frame. There is a general but not absolute correlation between the extent of ribosomal pausing and the protein levels found within the ATP synthase complex. We conclude that although it is not the sole factor, ribosome pausing may be a significant posttranscriptional mechanism affecting the expression of the large ATP synthase gene cluster in spinach chloroplasts.