Wheat seedling nuclease catalyzes the hydrolysis of intact, bihelical viral DNA or high molecular weight, native Escherichia coli DNA to produce limit polymers which are resistant to further hydrolysis by additional enzyme. These limit products are double-stranded polymers free of single strand interruptions and are terminated at their 5' ends with equal amounts of either deoxycytidylate or deoxyguanylate residues. The average size of the duplex limit products, as determined by (a) alkaline and neutral sucrose gradient sedimentation, (b) viscometric determination of molecular weight, and (c) 5'-end labeling, varies from 2 to 4 times 10-6 depending on the source of the DNA. The involvement of regions rich in adenine-thymine base pairs at the sites of cleavage of the DNA molecule is suggested by the following experimental results: (a) the copolymeric duplex, poly(dA-dt) is hydrolyzed at a rate comparable to that found for denatured calf thymus DNA, a rate which is several orders of magnitude faster than that at which native calf thymus DNA is hydrolyzed; (b) lambda DNA, which contains an adenine-thymine-rich region near its center, is rapidly cleaved to yield two fragments of similar size; (c) the rate of hydrolysis of native DNA is increased approximately 14-fold by increasing the reaction temperature from 20 degrees to 30 degrees.