We have isolated recombinant plasmids that contain cDNA inserts complementary to mRNAs encoding six different r-proteins of Dictyostelium discoideum. Southern and quantitative dot blot analyses have shown that each of the r-protein genes represented in these plasmids is encoded by a single copy gene and that these genes are not tightly linked to each other. We have determined the relative amount of the six r-protein mRNAs present in cells at intervals throughout development and find that for the first 9 hours of development, each of the mRNAs remains present at virtually the same level as in vegetatively growing cells. Between 9 and 11 hours of development, there is a rapid loss of these mRNAs to 15% or less of vegetative levels, and that low level remains, or slightly declines, through the late stages of development. We have shown that two post-transcriptional events contribute to the developmental regulation of the expression of the r-protein genes. The first involves a specific block to translational initiation that is not the result of inactivation of these mRNAs by decapping or deadenylation. The second is a change in the stability of these mRNAs during early development. In order to begin to analyze the role of specific sequences that may act as targets or signals in these events, we have cloned and sequenced a 1.9-kb genomic DNA fragment that encodes one of the r-proteins. We find that transcription of this gene begins in a pyrimidine-rich region that is not preceded by a TATA box, the gene contains a single intron of 350 bp, and there are two alternative 3' processing sites. In addition, the 5'-untranslated region of the transcript contains an unusually high percentage of G and C residues relative to other Dictyostelium mRNAs.