Restriction endonucleases isolated from Hemophilus influenzae, Hemophilus parainfluenzae, and Hemophilus aegyptius were used to cleave varphiX174 replicative form DNA into three sets of specific DNA fragments. The order of these fragments in the varphiX replicative form molecule was determined by (1) analysis of partial digest products, (2) analysis of overlapping sets of fragments produced by two different restrictive enzymes. On the basis of these results, a detailed physical map of the varphiX174 genome has been constructed with respect to the cleavage sites of all three enzymes.