The cytochrome P450 enzymes involved in xenobiotic metabolism are an excellent starting point for the directed evolution of novel biocatalysts due to their wide substrate specificity. A shuffled library of three highly homologous mammalian genes (for P450 2C9, P450 2C11 and P450 2C19) was constructed by applying a modified DNA family shuffling procedure. The modifications made to the traditional DNA shuffling protocols involved non-random digestion via the use of different combinations of restriction enzymes (REs) followed by isolation of fragments under 300 bp by size-selective filtration. Shuffled cytochrome P450 mutants were co-expressed in Escherichia coli with their redox partner, NADPH-cytochrome P450 reductase (NPR). We report here how non-random fragmentation may help in chimeragenesis within the areas of low sequence similarity such as substrate recognition sites (SRSs) that are generally underrepresented in recombination using the random fragmentation process. Size-selective filtration was used to limit recovery of incompletely digested fragments and consequently minimize the chances for contamination of the shuffled library with parental forms. No parental forms could be detected in the shuffled library using restriction fragment length polymorphism (RFLP) analysis, suggesting the library was free of parental contamination. Sequencing of randomly selected mutants demonstrated a high level of chimeragenesis with on average of 8.0+/-2.2 crossovers and a low level of mutagenesis with 5.2+/-2.8 spontaneous mutations per approximately 1.5 kbp of the full-length P450 sequence. The proportion of properly folded protein as indicated by the observation of characteristic Fe(II).CO vs. Fe(II) difference spectra was 15% (4/27) of analysed mutants. Screening of the shuffled library for indole oxidation revealed four clones with similar or higher levels of indigo pigment production to those of the parental P450s and two clones with elevated P450 expression. In this paper we present a method for the effective family shuffling of cytochrome P450 enzymes, applicable to the creation of mutant libraries with expanded metabolic diversity and with a significant proportion of functional clones.