The origin of DNA replication of bacteriophage f1 contains a nucleotide sequence that is used both for the initiation of viral (plus) strand synthesis and for its termination. With chimeric plasmids containing two f1 functional origins in the same orientation, synthesis of chimeric plus-strand DNA is initiated, after f1 infection, at either one of the two f1 origins and is terminated at the other. Thus, the chimeric plasmids segregate into two replicons, each of them containing only one f1 origin. This system has been used to test several fragments of the f1 origin varying in size or in nucleotide sequence for their ability to function in either initiation or termination of viral strand synthesis. Our data show that the f1 origin is composed of two overlapping but distinct domains (signals), one for initiation and the other for termination of plus-strand synthesis.