An in vitro system for bacteriophage Mu DNA replication using lysates on cellophane discs is described. Mu replication was monitored by DNA hybridization. Using a thermoinducible Mu lysogen, 30-50% of all DNA synthesis in vitro was Mu-specific. Mu DNA synthesis is semidiscontinuous. In the presence of the DNA ligase inhibitor NMN, about one-half of the DNA was in Okazaki pieces and one-half in large DNA. The Mu Okazaki pieces hybridized mainly to the Mu light strand; the large DNA hybridized mainly to the Mu heavy strand. Okazaki pieces isolated from uninfected cells also hybridized to 2000-3000 bases of host DNA present in Mu-separated strands. However, the host Okazaki pieces hybridize to both Mu strands symmetrically. Most, if not all, host sequences were represented in mature Mu viral DNA. The in vitro data are most consistent with models in which Mu sequences, oriented randomly in both directions in the host chromosome, have recruited a bacterial replisome which traverses the Mu genome from left to right.