The high cost of nicotinamide cofactors requires that they be regenerated in situ when used in preparative enzymatic synthesis. Numerous strategies have been tested for in situ regeneration of reduced and oxidized cofactors. Regeneration of reduced cofactors is relatively straightforward; regeneration of oxidized cofactors is more difficult. This review summarizes methods for preparation of the cofactors, factors influencing their stability and lifetime in solution, methods for their in situ regeneration, and process considerations relevant to their use in synthesis.