Abstract Heteroduplex replicative form of bacteriophage φX174 was prepared by annealing minus strand isolated from wild-type φX RF † † Abbreviation used: RF, replicative form. with plus strand DNA, obtained from temperature-sensitive and amber mutants of φX in different cistrons. Three different kinds of bursts were produced by spheroplasts infected with heteroduplex RF: bursts with only wild-type phage, bursts with only mutant phage and bursts with wild-type and mutant phage. The frequencies of these bursts for RF with heteroduplex regions in different cistrons were determined in single-burst experiments under permissive conditions. A gradient in the percentage of spheroplasts producing both genotypes, depending on the position of the mutation in the plus strand of heteroduplex RF on the genetic map of φX phage, was observed. The direction of replication of double-stranded φX DNA as well as the origin of replication were deduced from this gradient.