Members of the Spec gene family are expressed during embryonic development of the sea urchin, Strongylocentrotus purpuratus. The family encodes proteins related to the calmodulin/troponin C/myosin light chain group of calcium binding proteins and one gene, Spec1, has been studied extensively in our laboratory. In this paper, we analyze other members of the family, collectively termed Spec2 genes. We make use of several hybridization probes derived from Spec1 and Spec2 cDNA clones, which recognize different members of the family. Genomic DNA gel blot and slot blot analyses show that there are approximately eight Spec genes in the S. purpuratus genome. The structures of three Spec2 genes, Spec2a, Spec2c and Spec2d, are described. A 60 kb (kb = 10(3) bases or base-pairs) region of the genome contains the linked Spec1-Spec2c genes and two separate 20 kb regions contain the Spec2a and Spec2d genes. Six members of a repetitive sequence family are dispersed at various locations among the genes. The transcriptional initiation sites of the three Spec2 genes are mapped, and 400 to 500 base-pairs of 5'-flanking DNA sequenced. All three Spec2 genes initiate transcription approximately 120 base-pairs upstream from the 3' end of the first exon. In contrast, the 5' end of the Spec1 transcript begins about 107 base-pairs farther upstream, so it contains 5' untranslated sequences that correspond to non-transcribed 5'-flanking sequences of the Spec2 genes. There is little similarity among the sequences upstream from the CAP site of the Spec2 genes except the TATA consensus sequence and a repeating trinucleotide, AAC. Measurements of Spec mRNA levels during embryogenesis show that Spec1 mRNA begins to accumulate at the early blastula stage and is the most abundant; Spec2a/Spec2c mRNAs begin accumulating several hours later at the late blastula-early gastrula stage and reach about 40 to 60% the levels of Spec1; and Spec2d mRNAs accumulate mostly during the gastrula and pluteus stages with levels reaching only 2% those of Spec1. In situ hybridization with probes that recognize either all Spec2 mRNAs or only Spec2d mRNAs show that, like Spec1, these mRNAs are restricted to aboral ectoderm cells and their precursors. The Spec gene family represents a group of related genes whose mRNAs all accumulate in the same cell type but at different times and to different levels during embryogenesis.