The frequency of single nucleotide polymorphisms (SNPs) in downstream signaling proteins was determined by combination heteroduplex HPLC and double-stranded sequencing of genomic DNA from 96-144 congestive heart failure (CHF) patients. Analysis of 56 coding exons in 9 signaling genes revealed 17 novel and 8 previously reported synonymous (no change in amino acid) SNPs, as well as one novel nonsynonymous SNP in the Rad small G protein. Because this initial analysis failed to detect numerous SNPs reported in the NCBI and Celera databases, double-strand sequencing of relevant exons from 74-91 CHF patients was used to confirm the absence of 10 previously reported nonsynonymous SNPs. Our results show that synonymous SNPs are frequent in signaling protein genes, whereas nonsynonymous SNPs are rare, suggesting a high degree of evolutionary conservation among these downstream signaling molecules. Comparisons of our results to the NCBI and Celera databases indicates that 56% of their SNP entries are not detected in our cohort. Importantly, while 31% of database SNPs were verified, 69% of SNPs detected in our cohort are not included in these databases. These findings indicate that caution may be warranted in relying exclusively on SNP databases as catalogs for polymorphic signaling protein genes.