The D9 and D10 proteins of vaccinia virus are 25% identical to each other, contain a mutT motif characteristic of nudix hydrolases, and are conserved in all sequenced poxviruses. Previous studies indicated that overexpression of D10 and, to a lesser extent, D9 decreased the levels of capped mRNAs and their translation products. Here, we further characterized the D10 protein and showed that only trace amounts are associated with purified virions and that it is expressed exclusively at late times after vaccinia virus infection. A viable deletion mutant (vdeltaD10) produced smaller plaques and lower virus yields than either wild-type virus or a D9R deletion mutant (vdeltaD9). Purified vdeltaD10 virions appeared normal by microscopic examination and biochemical analysis but produced 6- to 10-fold-fewer plaques at the same concentration as wild-type or vdeltaD9 virions. When 4 PFU per cell of wild-type or vdeltaD9 virions or equal numbers of vdeltaD10 virions were used for inoculation, nearly all cells were infected in each case, but viral early and late transcription was initiated more slowly in vdeltaD10-infected cells than in the others. However, viral early transcripts accumulated to higher levels in vdeltaD10-infected cells than in cells infected with the wild type or vdeltaD9. In addition, viral early and late mRNAs and cellular actin mRNA persisted longer in vdeltaD10-infected cells than in others. Furthermore, analysis of pulse-labeled proteins indicated prolonged synthesis of cellular and viral early proteins. These results are consistent with a role for D10 in regulating RNA levels in poxvirus-infected cells.