Three Acinetobacter calcoaceticus transformation-deficient mutants, obtained by insertional mutagenesis with the nptll gene, have been characterized physiologically. One mutant (AAC211) was found to be completely transformation deficient, while two others, AAC213 and AAC214, were severely impaired in transformation efficiency (100-1000 times lower than the wild type). The latter applied to both chromosomal as well as plasmid DNA. Analysis of the chromosomal DNA fragments flanking the nptll gene in the mutants showed that mutants AAC213 and AAC214 had an insertion of the nptll gene in the same chromosomal region, but that they were the result of two independent mutational events, whereas the insertion in mutant AAC211 was at a different position. None of the three mutants showed phenotypic or genotypic characteristics typical of a RecA-deficient strain.