We have introduced four point mutations into the 5' junction (GTATGT) and TACTAAC box of a yeast intron-containing gene coding for beta-galactosidase. To analyze quantitatively mutant combinations, we deliberately avoided nucleotides adjacent to the sites of cleavage (GTATGT) and lariat formation (TACTAAC) and chose positions with expected weak effects on splicing. All four mutants do not affect, or only marginally affect, gene expression. The levels of precursor RNA and intermediates indicate, however, that splicing efficiency is affected in all cases; the first stage of splicing, 5' cleavage and lariat formation, is reduced two to 15-fold in these four mutants. The mutants were combined to generate compensatory and noncompensatory double mutant combinations. No evidence for a specific base-pairing interaction between the 5' junction and TACTAAC box could be obtained. The results suggest that all conserved positions in the 5' junction and TACTAAC box play a role in splicing efficiency.