Splice site nucleotide substitutions can be analyzed by comparing the individual information contents (R i , bits) of the normal and variant splice junction sequences . In the present study, we related splicing abnormalities to changes in R i values of 111 previously reported splice site substitutions in 41 different genes. Mutant donor and acceptor sites have significantly less information than their normal counterparts. With one possible exception, primary mutant sites with <2.4 bits were not spliced. Sites with R i values Β³2.4 bits but less than the corresponding natural site usually decreased, but did not abolish splicing. Substitutions that produced small changes in R i probably do not impair splicing and are often polymorphisms. The R i values of activated cryptic sites were generally comparable to or greater than those of the corresponding natural splice sites. Information analysis revealed preexisting cryptic splice junctions that are used instead of the mutated natural site. Other cryptic sites were created or strengthened by sequence changes that simultaneously altered the natural site. Comparison between normal and mutant splice site R i values distinguishes substitutions that impair splicing from those which do not, distinguishes null alleles from those that are partially functional, and detects activated cryptic splice sites.