To investigate gene expression from the Autographa californica nuclear polyhydrolysis virus genome (AcNPV), complementary DNA (cDNA) was synthesized from polyadenylated RNA transcribed at 2 h and 10 h postinfection (p.i.) and then cloned into Escherichia coli using plasmid pUC-9. Eighteen 2 h cDNA plasmids were homologous to five distinct regions of the viral genome, while forty-nine 10 h cDNA plasmids were homologous to 15 regions including the five regions transcribed at 2 h. Temporal expression of polyadenylated RNA transcribed from diverse regions of the genome was examined using Northern blot hybridization with the above 2 and 10 h cDNA probes. All regions displayed overlapping sets of RNAs. In addition to HindIII-I/EcoRI-F (IF) and HindIII-B2/EcoRI-H (B2H), several, but not all, regions showed a sequential appearance of higher molecular weight RNAs as the infection progressed. Each overlapping set of RNAs exhibited unique characteristics including variations in the number of overlapping transcripts, their temporal regulation, and their relative abundance during the course of infection.