A nuclease with novel activities has been isolated and purified to apparent homogeneity from pea chloroplasts. The enzyme prefers single-stranded (ss) circular DNA; its activity being 1500-fold higher with the ss circular DNA than with the linear double-stranded DNA substrates. The single-stranded DNase activity is stable at moderately high temperature (50 °C) and inhibited in the presence of 75 mM NaCl. It binds negatively supercoiled DNA in the stoichiometric fashion, but behaves catalytically on the single-stranded circular DNA. Although the DNase activity does not recognize any specific nucleotide sequence, the co-operative mode of activity seems to be a novel one. The protein is a monomer of 35 kDa and binds with DNA predominantly through electrostatic interactions. The drug distamycin blocks the endonuclease activity suggesting that the protein binds at the minor groove of DNA. A RNase activity has also been found associated with the ss-DNA endonuclease.