Ancient cells must have possessed small gene content. Primitive enzymes may have possessed broad specificity and undeveloped regulation mechanisms. The considerable substrate ambiguity of these enzymes resulted in the formation of minor amounts of erroneous products. Fortuitous formation of metabolites offered ancient cells maximum biochemical flexibility with minimal gene content. Gene duplication provided the opportunity for increased gene content and increased specialization of the diverging enzymes, the substrate specialization being further reinforced by the development of regualtory mechanisms. Recuritment of enzymes for new pathways did not necessarily require the sequential and backwardly evolving progression of evolutionary steps required by the hypothesis of retrograde evolution of biochemical pathways. Substrate ambiguity remains a conspicuous feature of many contemporary proteins, and evolutionary exploitation of substrate ambiguity in a variety of organisms is still apparent.