The characteristics of the soybean leghemoglobin lba gene promoter were analyzed and important promoter elements from the lba and lbc3 promoters were compared using transgenic Lotus corniculatus plants. A 5' deletion analysis of the lba promoter delimited two cis-acting elements controlling expression: a distal positive element (-1254, -884) required for expression and a proximal element (-285, -60) essential for full-level activity. In contrast to the corresponding region of the lbc3 promoter, the lba proximal element is unable to control expression from the heterologous CaMV 35S enhancer. The upstream positive element of the lba gene contains a position- and orientation-independent enhancer between positions (-1091, -788). The sequence of this enhancer region is conserved in the lbc3 gene upstream (-1333, -1132) of the previously assigned strong positive element (SPE; -1090, -947). The present analysis revealed some of the properties of this extended lbc3 SPE element. The extended element (-1364, -947) functions in both orientations from 5' locations whereas the SPE2 subcomponent (-1364, -1154) containing the conserved sequence is only active in the correct orientation. Removal of the SPE2 by internal deletion demonstrates that the SPE2 subcomponent is indispensable for the activity of the lbc3 upstream positive element. These results indicate that the upstream positive elements of the lba and lbc3 genes possess different properties although their conserved minimal enhancer sequence has similar function. This may reflect the differential expression of the two lb genes of Glycine max L.