To investigate the role of arginine in the folding of d-aminoacylase, seven arginine residues, R26, R152, R296, R302, R354, R377, and R391, among twelve arginine residues highly conserved in d-aminoacylase, N-acyl-d-aspartate amidohydrolase (d-AAase), and N-acyl-d-glutamate amidohydrolase (d-AGase) from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) were substituted with lysine by site-directed mutagenesis. The mutants, R26K, R152K, R296K, and R302K were identified as mutations that increase partitioning of the enzyme into inclusion bodies. No mutants with substitutions within the carboxyterminal segment were found to increase partitioning into inclusion bodies (R354K, R377K, and R392K). These results suggest that arginine residues that position between the N-terminus and central region can play an important role in facilitating folding or stabilizing the structure of d-aminoacylase. By anaerobic cultivation, the production level of R302K in the soluble fraction was improved. Coexpression of the DnaK-DnaJ-GrpE chaperone assisted the folding of R302K, and reduced the effect of the aeration conditions on the solubility of R302K. We hypothesized that R302K requires a larger amount of chaperones for efficient folding than the wild type enzyme.