The fitness effects of synonymous mutations, nucleotide changes that do not alter the encoded amino acid, have often been assumed to be neutral, but a growing body of evidence suggests otherwise. We used site-directed mutagenesis coupled with direct measures of competitive fitness to estimate the distribution of fitness effects among synonymous mutations for a gene under directional selection and capable of adapting via synonymous nucleotide changes. Synonymous mutations had highly variable fitness effects, both deleterious and beneficial, resembling those of nonsynonymous mutations in the same gene. This variation in fitness was underlain by changes in transcription linked to the creation of internal promoter sites. A positive correlation between fitness and the presence of synonymous substitutions across a phylogeny of related Pseudomonads suggests these mutations may be common in nature. Taken together, our results provide the most compelling evidence to date that synonymous mutations with non-neutral fitness effects may in fact be commonplace.

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