Two substitutions were made for Arg41 in the polar loop of subunit c of the Escherichia coli F1F0 H(+)-transporting ATP synthase. The R41K and R41H mutants were initially studied by use of a plasmid carrying the complete c R41K or c R41H unc (F1F0) operon in a chromosomal strain deleted for the unc operon. The extent of F0 incorporation into membranes of these cells was quite variable, and the system was concluded to be unsuitable for biochemical characterization. Ultimately, the mutant genes were recombined into the chromosome using a novel method for the unc system. The biochemical phenotype of the chromosomally expressed mutants proved to be reproducible. The c R41H mutation causes a specific defect in assembly of F0, i.e. subunit a was not incorporated into the membrane despite near normal incorporation of subunits b and c. On the other hand, c R41K mutant F0 assembled normally in one of two background strains studied. (In the second genetic background, subunit a was inefficiently incorporated into the c R41K membrane.) In membranes prepared from a c R41K strain assembling a complete F0, R41K F0 was found to bind F1 with near normal affinity and to transport H+ at near normal rates. Although R41K F0 binds F1, F1-ATPase activity and H+ transport remained uncoupled. The uncoupling was indicated by a lack of ATP-driven H+ translocation and by the high proton permeability of membranes with F1 bound to F0. The uncoupled phenotype of the R41K mutant closely resembles that previously reported for the c Q42E mutant.