Random mutagenesis and selection using phage or cell surface display provides an efficient method for affinity maturation of single chain Fv (scFv) antibodies, thereby improving function in various applications. To investigate the effects of mutation frequency on affinity maturation, error-prone PCR was used to generate libraries containing an average (m) of between 1.7 and 22.5 base substitutions per gene in a high affinity scFv antibody that binds to the cardiac glycoside digoxigenin. The scFv antibody libraries were displayed on Escherichia coli, and mutant populations were analyzed by flow cytometry. At low to moderate mutation frequencies with an average mutation rate of m </= 8, the fraction of clones exhibiting binding to a fluorescently labeled conjugate of digoxigenin decreased exponentially (r(2) = 0.99), but the most highly mutated library (m = 22.5) had significantly more active clones than expected relative to this trend. A library with a low error rate (m = 1.7), one with moderate error rate (m = 3.8), and the one with high error rate (m = 22.5) were screened for high affinity clones under conditions of identical stringency using fluorescence-activated cell sorting. After several rounds of enrichment, each of the three libraries yielded clones with improved affinity for the hapten. The moderate and high error rate libraries gave rise to clones exhibiting the greatest affinity improvement. Taken together, our results indicate that (i) functional clones occur at an unexpectedly high frequency in hypermutated libraries, (ii) gain-of-function mutants are well represented in such libraries, and (iii) the majority of the scFv mutations leading to higher affinity correspond to residues distant from the binding site.