Cryptic splice sites are used only when use of a natural splice site is disrupted by mutation. To determine the features that distinguish authentic from cryptic 5' splice sites (5'ss), we systematically analyzed a set of 76 cryptic 5'ss derived from 46 human genes. These cryptic 5'ss have a similar frequency distribution in exons and introns, and are usually located close to the authentic 5'ss. Statistical analysis of the strengths of the 5'ss using the Shapiro and Senapathy matrix revealed that authentic 5'ss have significantly higher score values than cryptic 5'ss, which in turn have higher values than the mutant ones. beta-Globin provides an interesting exception to this rule, so we chose it for detailed experimental analysis in vitro. We found that the sequences of the beta-globin authentic and cryptic 5'ss, but not their surrounding context, determine the correct 5'ss choice, although their respective scores do not reflect this functional difference. Our analysis provides a statistical basis to explain the competitive advantage of authentic over cryptic 5'ss in most cases, and should facilitate the development of tools to reliably predict the effect of disease-associated 5'ss-disrupting mutations at the mRNA level.