This study describes the investigation of the possibility of genetic transformation of already immobilized competent cells by plasmids. The preliminary prepared competent cells were entrapped into granules of an insoluble carrier, a cryogel of poly(vinyl alcohol). The specific activity of organophosphatehydrolase and ampicillin resistance conferred by pOPf1 plasmid were used as markers of successful transformation of the immobilized competent cells. The effect of main experimental conditions of transformation usually used for free cells, i.e., time of incubation of cells with DNA solution, temperature, and time of heat shock, on the transformation efficiency of the immobilized competent cells has been studied. A number of important factors of preparation of immobilized transformed cells, i.e., the concentration of immobilized competent cells inside the granules, the concentration of DNA solution used for transformation, have been shown to affect the OPH-activity of the final immobilized transformants. The possibility of transformation of the immobilized competent cells by both single- and double-stranded plasmid DNA molecules has been demonstrated.