Zein cDNA clones were used to study the organization of zein genes within the genome of the inbred maize W64A. When individual clones for the two larger molecular-weight classes of zein proteins (Mr = 22,000; Mr = 19,000) were used as probes for Southern blot hybridizations of genomic DNA, multiple restriction fragments were found to hybridize. Reconstruction analyses using moderately stringent criteria were used to estimate a total of 70-80 zein sequences within the genome of this inbred maize. The hybridization patterns suggest that zein sequences are clustered within the same restriction fragment. When criteria permitting less cross-hybridization of homologous sequences (Tm - 10 degrees C) were used, the banding pattern changed, with some of the bands being reduced in intensity or eliminated entirely. Therefore, by control of hybridization criteria, particular zein genes may be more readily distinguished in a Southern blot analysis. The Southern blot hybridization pattern for the Mr = 15,000 zein was less complex. Only a single major band was found, with sufficient hybridization intensity for two or three genes. Genomic Southern analyses of other inbred maizes and related grasses showed similarly complex hybridization patterns with cDNA probes for the 19,000- and 22,000-molecular-weight zeins, suggesting that these sequences have been conserved over evolutionary time. The zein multigene family may therefore have arisen by gene duplication before divergence of the maize, teosinte, and Tripsacum species from a common ancestor.