We have engineered a mutant form of Escherichia coli F1-ATPase which is tryptophan-free and contains five mutations, namely δW28L/αW513F/γW108Y/γW206Y/ βW107F. A strain carrying all five mutations grew normally by oxidative phosphorylation. Purified mutant F1-ATPase showed V(max) and K(m) both 65% higher than wild-type, resulting in k(cat)/K(m) the same as wild-type. The pH dependence of ATPase activity in mutant enzyme was very similar to that in wild-type. Catalytic-site nucleotide-binding characteristics were measured using the analog lin-benzo-ADP and sensitivity to inhibitors was tested using dicyclohexylcarbodiimide, azide and aurovertin. The mutant enzyme was very similar to wild-type in each of these characteristics. The fluorescence spectrum of mutant enzyme confirmed the absence of tryptophan. We have therefore established that it is possible to generate a tryptophan-free enzyme which retains normal catalytic function, oligomeric stability and in vivo assembly.