Bacteriophage phi6 has a double-stranded RNA genome composed of three linear segments, L, M, and S. The innermost particle in the virion of phi6, like in the other dsRNA viruses, is an RNA-dependent RNA polymerase complex, which carries out all the functions needed for the replication of the viral genome. Empty polymerase complexes can package the single-stranded copies of the viral genome segments, replicate the packaged segments into double-stranded form (minus strand synthesis), and then produce new plus strands (transcripts) from the double-stranded RNA templates. The three viral genomic segments contain unique packaging signals at their 5' ends, and minus strand synthesis initiation is dependent on the sequence at the 3' end. Here we have constructed chimeric segments that have the packaging signal from one segment and the minus strand synthesis initiation signal from another segment. Using purified recombinant polymerase complexes and single-stranded/chimeric and original RNA segments, we have analyzed the packaging and replication regulation operating in in vitro conditions. We show that the 5' end of the L genome segment in single-stranded form is needed to switch from the packaging to the minus strand synthesis and the same sequence is required in double-stranded form to switch on plus strand synthesis. In addition we have constructed deletions to the M segment to analyze the possible regulatory role of the internal noncoding area of this segment.