Site-directed mutagenesis has been used to study the effects of mutations resulting in surface charge changes near the Fe(Cys) 4 site of Clostridium pasteurianum rubredoxin (Rd). As predicted by simple electrostatics considerations, Rd variants with positively charged arginine residues in place of neutral surface residues ([V8R] and [L41R]) exhibit significant increases in the Fe(II/III) reduction potential. Contrary to electrostatics predictions, [V8D] and [V41D] Rd variants also exhibit significant increases in the Fe(II/III) reduction potential. These results indicate that protein electrostatic effects do not dominate as determinants of metal-site reduction potential in C. pasteurianum Rd. A hypothesis is developed that increased solvent accessibility and the resultant increase in polarity of the Fe(Cys) 4 site dominate as determinants of the reduction potential in this protein. Possible experimental tests of this hypothesis are discussed.