S1 nuclease protection and primer extension analyses were used to determine the 5' end of the Autographa californica nuclear polyhedrosis virus 603 open reading frame (ORF) transcript upstream of and on the opposite strand to the polyhedrin gene. These analyses suggested that the 5' end of the 603 ORF was located near the initiation site for polyhedrin gene transcription. Primer extension products of reverse transcription of 603 RNA were dependent on the presence of the TAAG sequence, an essential polyhedrin promoter element. The results could be interpreted as indicating bidirectional transcription from the TAAG element. However, the data could also be due to an artefact of antisense transcripts and RNA duplex formation in this region. Since the bidirectional transcriptional model is not consistent with other mapping data and Northern blot analysis, we conclude that the presence of antisense RNA can result in mapping artefacts and that caution must be taken in interpreting data where overlapping sense and antisense RNAs are present.