Intragenic recombination rapidly creates protein sequence diversity compared with random mutation, but little is known about the relative effects of recombination and mutation on protein function. Here, we compare recombination of the distantly related -lactamases PSE-4 and TEM-1 to mutation of PSE-4. We show that, among -lactamase variants containing the same number of amino acid substitutions, variants created by recombination retain function with a significantly higher probability than those generated by random mutagenesis. We present a simple model that accurately captures the differing effects of mutation and recombination in real and simulated proteins with only four parameters: (i) the amino acid sequence distance between parents, (ii) the number of substitutions, (iii) the average probability that random substitutions will preserve function, and (iv) the average probability that substitutions generated by recombination will preserve function. Our results expose a fundamental functional enrichment in regions of protein sequence space accessible by recombination and provide a framework for evaluating whether the relative rates of mutation and recombination observed in nature reflect the underlying imbalance in their effects on protein function. directed evolution ͉ mutagenesis ͉ neutrality ͉ lattice proteins ͉ site-directed recombination A major goal in understanding the molecular basis of evolution is to quantitatively describe how effectively mutation and recombination traverse protein sequence space to create new functional proteins (1). Protein sequence distance (measured by counting the number of amino acid substitutions, m, separating two sequences) is a fundamental metric of evolutionary rate and relationships (2), diversity of structure and function (3), and a key variable in protein engineering (4, 5), whereas mutation and recombination are its biochemical cause. Genetic studies (6, 7) and algorithmic inferences from biological sequence data (8-10) have revealed that recombination can occur preferentially within coding sequences, at times with a higher frequency than mutation (11, 12). When sequences encoding divergent but related proteins recombine, large distances may be traveled in sequence space relative to random mutation (13-16) without disturbing function and͞or structure. However, a complete understanding of the underlying relative efficiency of mutation and recombination in accessing nearby or distant regions of sequence space cannot be gained from genomic sequences because these become available only after natural selection has acted.Laboratory (17) and in silico (18) evolution experiments, in contrast, can be used to quantitatively differentiate the effects of mutation or recombination on protein structure and function. By screening or selecting libraries of proteins for retention of parental function and determining the sequences of functional and nonfunctional proteins, one can determine how the retention of function or structure depends on m, the sequence distance. This type of ana...