We have analyzed the expression and function of the intron-encoded bI4 maturase when frame-shift mutations in the upstream exon alter the translational process. By constructing secondary cis-acting mutations within the bI4 intron, we observed (1) that the bI4 maturase is still translated in the presence of the upstream mutation, albeit in very low amounts, and (2) that the limited amounts of bI4 maturase made under these conditions is no longer able to promote the splicing process of the aI4 intron. These observations, which further strengthen the maturase model, strongly suggest that bI4 maturase acts sequentially on the bI4 intron and then on the aI4 intron.