Two infectious drug-resistance (R) factors, R28K and R6K, each conferring resistance to a number of penicillins by the synthesis of a penicillinase, were transferred to Proteus mirabilis PM1 and Escherichia coli RC85 host strains. Deoxyribonucleic acid (DNA) extracted from these strains was separated by density-gradient centrifugation and subjected to electron microscopy by use of a modification of the protein-monolayer diffusion technique. Analytical density-gradient centrifugation of the purified DNA from PM1 strains showed, in addition to the major peak at a density of 1.698 g/cm(3) characteristic of Proteus chromosomal DNA, a single satellite band at a density of 1.710 g/cm(3) [guanine plus cytosine (GC) base ratio 50%] for R28K and at 1.704 g/cm(3) (GC base ratio 45%) for R6K. Direct CsCl density-gradient centrifugation of crude lysates of the E. coli (R28K)(+) strain in the presence of ethidium bromide gave rise to a sedimentation profile with a single satellite peak containing covalently closed circular (CCC) DNA molecules with a mean contour length of 21.4 mum [44 x 10(6) atomic mass units (AMU)], although a minority was 13.6 mum in length. From the size of the major class, it was estimated that there were two to three copies of the R28K factor present as CCC molecules per chromosome at various phases of cellular growth. Similar studies of the E. coli (R6K)(+) lysates showed two satellite peaks; peak I contained mostly CCC molecules of contour length 12.8 mum (26 x 10(6) AMU), and peak II, intermediate to peak I and the chromosomal peak, contained CCC molecules of a similar size, together with about equal numbers of catenated molecules, mostly dimers consisting of two interlocked monomers of 12.8 mum. A smaller number (ca. 0.1%) of higher catenanes was also seen. The number of CCC copies of the R6K factor per chromosome present in peak I was calculated as 13 at logarithmic phase and 38 at stationary phase. In peak II, a constant ratio of about one catenated dimer per chromosome was found at all phases of growth. Penicillinase assays of cultures at different phases of growth showed a correlation between the estimated number of R-factor copies present as CCC molecules and the enzyme activity per cell for both R28K and R6K.