The SpMTA metallothionein gene of the sea urchin Strongylocentrotus purpuratus is regulated developmentally, histospecifically, and by heavy-metal induction. The sequenced 5' flank of the gene can be divided into proximal, middle, and distal regions, each containing a pair of metal response elements (MREs). Canonical 7-bp core sequences are present in all except the middle-region MREs c and d, which contain 1-bp mismatches. Metal-induced expression in transgenic blastulae was increased with each consecutive addition of the middle and distal regions to a chimeric reporter gene construct containing the proximal SpMTA promoter region. Reduced metal induction through point mutation of the distal MREs e and f indicated that the MREs themselves were largely responsible for the transcriptional increase. These activities were further enhanced by SpMTA intron 1, but not when a specific interior region of the intron had been deleted. The atypical MREs c and d did not support induction by themselves, i.e., when present alone with mutated proximal MREs a and b. However, in the presence of intron 1, they were able to substitute for the nullified MREs a and b in the promotion of metal-induced expression. This capability suggests, furthermore, that these atypical MREs, in addition to responding to an intron 1 region, participate cooperatively with the canonical proximal MREs.