The first two steps towards increasing ampicillin resistance in Escherichia coli concern the genes ampA and ampB which are located at least 20 min from each other (Eriksson-Grennberg et al. 1965 and the two preceding papers). This paper describes a third class of ampA-containing mutants (designated ampAB) which are resistant to D, L-ampicillin concentrations of 100 μg/ml. When such as F-strain (D31) was crossed to different Hfr strains analysis of the trp+ and proB+ recombinants indicated that resistance genes were located between trp and proB. Altogether five classes of recombinants were produced but only the two genes ampA and ampB were recovered. One interpretation suggested is that the resistance of D31 is due to the presence of ampA and ampB and a chromosomal aberration by which ampA was moved to a position near ampB. It was possible to transduce both intermediate levels of resistance as well as the full resistance of an ampAB donor strain, but the strains produced were unstable. In crosses the presence of ampAB in the recipient reduced the number of recombinants by reducing the number of stable pairs. In an Hfr strain ampAB was shown to give rise to additional difficulties in establishing the cell contact during mating. Some ampicillin resistant mutants also showed decreased ability to propagate the RNA phage MS2.