Pseudomonas stutzeri, in addition to being transformed by duplex DNA, is also transformed by the sense or antisense strand of the genetic marker employed (hisX+) or by heat-denatured chromosomal DNA. Transformation was absent in non-competent cells and in mutants defective for pilus biogenesis (pilA, pilC) and function (pilT) or DNA translocation into the cytoplasm (comA). Uptake of 3H-thymidine-labeled single-stranded DNA was hardly detectable reflecting the 20- to 60-fold lower transformation compared to duplex DNA. The results suggest that the steps in natural transformation also accommodate single-stranded DNA and that DNA translocation from the periplasm into the cytoplasm is not necessarily coupled to the degradation of a complementary strand. Small DNA single-stranded fragments are thus not excluded from horizontal gene transfer by transformation.