The expression of a soybean leghemoglobin 5'lbc3-GUS-3'nos chimeric gene was analyzed in Lotus corniculatus after site-specific mutagenesis of the nodulin consensus sequences, AAAGAT and CTCTT, present in the organ-specific element (OSE) (-139 to -102). Full-length promoters (-1956, +46) carrying clustered point mutations in the CTCTT sequence or in both the AAAGAT and the CTCTT sequences were inactive. Point mutations in the AAAGAT sequence had only minor effects on the expression level. Substitution of the ATTG sequence between the AAAGAT and the CTCTT sequences in the OSE reduced the activity in nodules to 10%. This, together with the conservation of the ATTGT sequence in the same position of leghemoglobin genes from other legumes, indicates that these sequences, in addition to the nodulin consensus sequences of the OSE, are important for high-level nodule-specific expression. Substitution of the CTCTT sequences outside the OSE (-44, -40 and -79, -75) results in promoter activities of approximately 50%.