Most data available from in vivo sources regarding the impact of somatic hypermutation on Ab V region structure and function are heavily biased due to the influence of clonal selection. In an effort to address this issue directly, we "randomly" introduced point mutations throughout the length of the VH region of an anti-p-azophenylarsonate (Ars) Ab expressed as an Fab in the phage display format. This was accomplished by means of an error-prone PCR with two protocols, which resulted in two mutant libraries. The nature of the nucleotide substitutions obtained from each protocol differed from each other and resulted in different frequencies of phage clones that did not appear to contain Fab on their surfaces. However, the majority of mutants in both libraries lacked detectable Fab expression. Screening of the library containing the most expressed Fabs for those that had gained affinity for structurally related haptens yielded two independent mutants that lacked detectable affinity for Ars and had high affinity for p-azophenylsulfonate. These mutants both contained amino acid substitutions from Asn to Ser or Thr at VH CDR1 position 35, a putative Ars contact residue. In this paper, we discuss the significance of these data with regard to the frequencies of V region loss of function, gain of increased affinity, and gain of altered specificity that result from somatic hypermutation in vivo.