The transcriptional organization of the Drosophila melanogaster serendipity (sry) locus (previously designated EH8) has been investigated by DNA sequencing, S1 nuclease mapping, and primer extension analysis. The data indicate that the five different (and partially overlapping) sry messenger RNAs detectable in early embryos are initiated at three separate sites, each directly upstream from one of the three protein-coding regions, designated (in 5' to 3' order) beta, alpha and delta. All of the sry mRNAs are transcribed in the same direction. The two blastoderm stage-specific sry mRNAs both include the alpha-coding region and have the same 5' terminus, only 183 nucleotides downstream from the 3' terminus of a beta region transcript (which is transcribed in ovaries). Similarly, only 331 nucleotides separate the 5' end of a delta region transcript from the major 3' end of the alpha region transcripts. One of the five sry embryonic mRNAs includes both the beta and alpha protein-coding segments and the spacer region in between, while another mRNA includes both the alpha and delta protein-coding regions as well as the spacer in between. The two read-through transcripts probably result from the failure to undergo a 3' cleavage and polyadenylation event rather than from differential splicing. As the three other sry embryo mRNAs are each transcribed from a single protein-coding region, it would appear that the alpha, beta and delta open reading frames correspond to three separate genes. Codon bias analysis reinforces the notion that these three genes code for bona fide proteins with translation starting at the first in-frame AUG codon. The predicted beta and delta polypeptides show partial amino acid sequence homology, suggesting a common evolutionary origin.