We have used a full-length cDNA clone of a mouse hepatitis virus strain A59 defective interfering (DI) RNA, pMIDI-C, and cassette mutagenesis to study the mechanism of coronavirus subgenomic mRNA synthesis. Promoter sequences closely resembling those of subgenomic mRNAs 3 and 7 were inserted into MIDI-C. Both subgenomic RNA promoters gave rise to the synthesis of a subgenomic DI RNA in virus-infected and DI RNA-transfected cells. From a mutagenic analysis of the promoters we concluded the following. (i) The extent of base pairing between the leader RNA and the intergenic promoter sequence does not control subgenomic RNA abundance. (ii) Promoter recognition does not rely on base pairing only. Presumably, transcription initiation requires recognition of the promoter sequence by the transcriptase. (iii) Fusion of leader and body sequences takes place at multiple--possibly random--sites within the intergenic promoter sequence. A model is presented in which, prior to elongation, the leader RNA is trimmed by a processive 3'-->5' nuclease.