A reconstituted transcription and RNA replication system for human parainfluenza virus type 3 (HPIV3) was developed using components expressed intracellularly from transfected plasmids driven by T7 RNA polymerase supplied by a vaccinia virus recombinant. The system is based on a negative-sense analog of HPIV3 genomic RNA in which the viral genes were deleted and replaced with that encoding bacterial chloramphenicol acetyl transferase (CAT). The N, P, and L proteins expressed from cotransfected plasmids were necessary and sufficient to direct efficient transcription and RNA replication. Transcription yielded subgenomic polyadenylated mRNA, which was isolated by oligo(dT) chromatography. RNA replication yielded a mini-antigenome and progeny minigenome, which were shown to be encapsidated based on resistance to digestion with micrococcal nuclease. A panel of cDNAs was constructed to encode minigenomes which differed in length by single-nucleotide increments. Transcription and RNA replication in the reconstituted system were most efficient for the minigenome whose length was an even multiple of six. Both RNA replication and transcription appeared to be governed by the rule. However, minigenomes whose lengths were one nucleotide greater than or less than an even multiple of six also were very active, especially in RNA replication, indicating that the rule was not absolute.