For the efficient study of replication of DNA, the cyanobacterium Synechocystis PCC 6803 was first permeabilized by either L-alpha-lysophosphatidylcholine (LPC) or lysozyme-EDTA treatment. Permeability of the treated cells was evidenced by the incorporation of exogenously added P-32-TTP into DNA. In cells permeabilized by treatment with either method, the P-32-TTP incorporation at 30 degrees C was appreciably higher than that in untreated control cells and increased with time for about 4 h. In addition, treated cells became permeable to proteins such as DNase I and micrococcal nuclease, which entered cells and degraded the newly synthesized DNA. Lysozyme/ EDTA-treated cells not only incorporated P-32-TTP more efficiently than did LPC-permeabilized cells, but were capable of uptake and synthesis of exogenously supplied cyanobacterial plasmids isolated from Synechocystis 6803. This capacity of lysozyme/EDTA-treated Synechocystis to catalyze replication of exogenous DNA will allow the facile identification of DNA replication origins and their related regulatory sequences.