Coronaviruses synthesize a nested set of six to eight subgenomic (sg) mRNAs in infected cells. These mRNAs are produced in different, but constant, molar ratios. It is unclear which factors control the different levels of sg mRNAs. To determine whether the intergenic sequence (IS) involved in sg mRNA synthesis could affect the transcription efficiencies of other ISs and in this way regulate transcription levels, we inserted multiple ISs at different positions into a mouse hepatitis virus defective interfering RNA. Quantitation of the sg RNAs produced by identical ISs in different sequence contexts led to the following conclusions: (i) transcription efficiency depends on the location of the IS in the defective interfering virus genome, (ii) downstream ISs have a negative effect on transcription levels from upstream ISs, and (iii) upstream ISs have little or no effect on downstream ISs. The observation that a downstream IS downregulates the amounts of sg RNA produced by an upstream IS explains why the smaller sg RNAs are, in general, produced in larger quantities than the larger sg RNAs. Our data are consistent with coronavirus transcription models in which ISs attenuate transcription. In these models, larger sg RNAs are synthesized in smaller amounts because they encounter more attenuating ISs during their synthesis.