Using øA replicative-form DNA (RF), the properties of the calcium-dependent trans-fection system were investigated in detail. Calcium ions seem to induce preferential uptake of double-stranded DNA by the treated bacteria, rather than causing nonspecific or irreversible increase in the cellular permeability. In this system, Mg2+ was ineffective as a substitute for Ca2+. Transfection does not take place when cells suitably pretreated with Ca2+ are mixed with DNA in media deficient in the cation. In 50mM CaCl2, viral DNA can infect competent cells at 0°C. The yield of transfectants decreased markedly if the Ca2+-treated bacteria and DNA were mixed at temperatures, higher than 20°C and plated directly. Efficient transfection, however, occurred when DNA-cell mixtures preincubated at higher temperatures were plated after quenching at 0°C. Cells pretreated with EDTA did not develop any competence upon subsequent Ca2+ treatment, suggesting possible involvement of surface lipopolysaccharide and/or lipoprotein in the interaction with DNA. Dialyzed supernatant obtained from EDTA-cell mixture inhibited the transfection. The Ca2+-dependent transfection is inhibited by low concentrations of phosphate and is very sensitive to ionic detergents such as. cetyltrimethylammonium bromide or sodium dodecyl sulfate. Double-stranded hetero-logous DNA considerably inhibits RF infection, while the inhibitory effect of single-stranded DNA or RNA is relatively small. The infection occurs in the presence of certain metabolic inhibitors such as dinitrophenol or sodium azide. These results suggest that the Ca2+-dependent uptake of DNA is physicochemical or at least energy-independent. The competence level of bacterial mutants deficient in phospholipase A was as high as that of wild-type cells. The effect of culture conditions such as growth phase or temperature was also examined. Based on these results, the mechanism of Ca2+-dependent competence was discussed with particular reference to liquid crystallization of the cell envelope lipids.