Soluble guanylyl cyclase (sGC) is activated upon the interaction of NO with heme in the sGC beta1 subunit. To identify the domains contributing to heme-binding, we constructed a series of deletion mutants of the beta1 subunit, and evaluated their heme-binding capability. Deletion mutants consisting of residues 1-120 [beta1(1-120)] and 80-385 [beta1(80-385)] were the shortest mutants exhibiting heme binding among the C-terminal and N-terminal-truncated mutants, respectively. The region common to both beta1(1-120) and beta1(80-385), i.e., residues 80-120, is therefore essential for heme binding, although the residues 341-385 play an auxiliary role in heme binding. Two deletion mutants, beta1(80-195) and beta1(60-195), which include only the essential region, exhibited strong heme binding and spectral properties similar to those of the nitrosyl complex of native sGC. Thus, these heme-binding core proteins may serve as model proteins for future studies on the tertiary structure of the nitrosyl complex of sGC.