The time course of the syntheses of Punta Toro (PT) phlebovirus (Bunyaviridae) small (S)-size viral RNA (S vRNA), viral complementary RNA (S vcRNA), and messenger RNA (S mRNA) species has been analyzed using single-stranded DNA probes representing the two S-coded gene products. The data obtained support the conclusion that PT S RNA has an ambisense coding strategy (T. Ihara, H. Akashi, and D. H. L. Bishop, Virology 136, 293-306, 1984) with the viral nucleocapsid protein, N, encoded in a viral-complementary, subgenomic, mRNA species and a putative nonstructural protein, NSs, encoded in a viral-sense, subgenomic, second S mRNA species. In the absence of puromycin (or cycloheximide) full-length S vRNA, S vcRNA, and subgenomic N mRNA and putative NSs mRNA species were identified in PT virus-infected cell extracts. In the presence of inhibitors of protein synthesis (puromycin or cycloheximide) newly synthesized N mRNA species were detected, but not full-length S vcRNA, nor S vRNA, nor the S coded NSs mRNA species. The mRNA species recovered from drug-treated cells have been translated in vitro to synthesize viral N protein. Analyses of the 5' ends of the N and NSs mRNA species have shown them to be heterogeneous in sequence and some 11-18 bases longer than the ends of the genomic RNA species, indicating that they represent nonviral primer sequences like those identified for bunyavirus mRNA species (D. H. L. Bishop, M. E. Gay, and Y. Matsuoka, Nucleic Acids Res. 11, 6409-6418, 1983). The presence of such additional sequences on mRNA derived from representatives of two Bunyaviridae genera appears by these analyses to be a more conserved feature than the S RNA coding arrangement of the respective viruses.