Dehydrins (LEA D11 proteins) are the products of multigene families in a number of higher plants. To date, however, only one dehydrin locus, dhn1 (a major embryo and drought-induced protein of ca. 18 kDa) has been placed on chromosome 6L of the genetic linkage map of maize. The presence of a larger, ca. 40 kDa embryo protein that is also specifically detected by anti-dehydrin antibodies had been observed in some maize inbreds, including B73, suggesting that other dhn loci may exist. The ca. 22 kDa and ca. 40 kDa immunopositive proteins were purified from B73 and their amino acid compositions determined. The two proteins' amino acid compositions are typical of dehydrins, yet they differ from each other, indicating that they are distinct dhn gene products. Different size alleles for both proteins, or presence/absence in the case of the ca. 40 kDa protein, were evident from comparisons of embryo proteins of various maize inbreds. Analysis of segregating F2 progeny derived from self-pollination of F1 hybrids from four crosses (B73 x OH43, Mo17 x A632, AHO x A632, Latente x A632) revealed that alleles of the two genes assort independently. Map positions of the two dhn loci were then determined using two maize recombinant inbred line (RIL) mapping populations. The predicted map position of the gene controlling production of the ca. 22 kDa protein confirmed that this protein is the product of the dhn1 gene. The gene encoding the ca. 40 kDa dehydrin-like protein maps to a new locus on chromosome 9S near wx1, which we have named dhn2.