The antibiotic resistance plasmid R1drd19Km- which has spontaneously lost its kanamycin resistance marker, and its derivative pON5300, were analysed using the restriction endonucleases SalI, BamHI, HindIII and EcoRI. The fragment patterns were compared with that of the R1drd19 and the fragments responsible for kanamycin resistance were found to be missing in R1drd19Km- and pON5300. In these plasmids a 7 Mg/mol EcoRI fragment was observed instead of the D (6.3 Mg/mol) fragment of R1drd19. Further a new 6 Mg/mol EcoRI restriction fragment was observed in pON5300. Using double digestions it was shown that the new fragment does not carry restriction sites for HindIII, BamHI and SalI endonucleases. The non-homology of the analysed plasmid was proved electron microscopically by heteroduplex techniques. The possibility of amplification in the regulatory region for the expression of R-determinants in pON5300 is discussed.