Two morphologically different Aspergillus parasiticus strains, one producing aflatoxins, abundant conidia but few sclerotia (BN9) and the other producing O-methyl-sterimatocystin (OMST), copious sclerotia but a low number of conidia (RH), were used to assess the role of crzA which encodes a putative calcium-signaling pathway regulatory protein. Under standard culture conditions, BN9 Delta crzA mutants conidiated normally but decreased slightly in radial growth, regardless of illumination conditions. RH Delta crzA mutants produced only conidia under light and showed decreased conidiation and delayed sclerotial formation in the dark. Regulation of conidiation of both A. parasiticus strains by light was independent of crzA. Increased concentrations of lithium, sodium, and potassium impaired conidiation and sclerotial formation of the RH Delta crzA mutants but they did not affect conidiation of the BN9 Delta crzA mutants. Vegetative growth and asexual development of both Delta crzA mutants were hypersensitive to increased calcium concentrations. Calcium supplementation (10 mM) resulted in 3-fold and 2-fold decreases in the relative expression of the endoplasmic reticulum calcium ATPase 2 gene in the BN9 and RH parental strains, respectively, but changes in both Delta crzA mutants were less significant. Compared to the parental strains, the Delta crzA mutants barely produced aflatoxins or OMST after the calcium supplementation. The relative expression levels of aflatoxin biosynthesis genes, nor1, ver1, and omtA, in both Delta crzA mutants were decreased significantly, but the decreases in the parental strains were at much lower extents. CrzA is required for growth and development and for aflatoxin biosynthesis under calcium stress conditions.