We analyzed cDNA libraries from developing endosperm of the B73 maize inbred line to evaluate the expression of storage protein genes. This study showed that zeins are by far the most highly expressed genes in the endosperm, but we found an inverse relationship between the number of zein genes and the relative amount of specific mRNAs. Although alpha-zeins are encoded by large multigene families, only a few of these genes are transcribed at high or detectable levels. In contrast, relatively small gene families encode the gamma- and delta-zeins, and members of these gene families, especially the gamma-zeins, are highly expressed. Knowledge of expressed storage protein genes allowed the development of DNA and antibody probes that distinguish between closely related gene family members. Using in situ hybridization, we found differences in the temporal and spatial expression of the alpha-, gamma-, and delta-zein gene families, which provides evidence that gamma-zeins are synthesized throughout the endosperm before alpha- and delta-zeins. This observation is consistent with earlier studies that suggested that gamma-zeins play an important role in prolamin protein body assembly. Analysis of endosperm cDNAs also revealed several previously unidentified proteins, including a 50-kD gamma-zein, an 18-kD alpha-globulin, and a legumin-related protein. Immunolocalization of the 50-kD gamma-zein showed this protein to be located at the surface of prolamin-containing protein bodies, similar to other gamma-zeins. The 18-kD alpha-globulin, however, is deposited in novel, vacuole-like organelles that were not described previously in maize endosperm.