The agnoprotein of simian virus 40 (SV40) is a 61-amino-acid protein encoded in the leader of some late mRNAs. In indirect immunofluorescence studies with antisera against SV40 capsid proteins, we show that mutants which make no agnoprotein display abnormal perinuclear-nuclear localization of VP1, the major capsid protein, but not VP2 or VP3, the minor capsid proteins. In wild-type (WT) SV40-infected CV-1P cells, VP1 was found predominantly in the cytoplasm until 36 h postinfection (p.i.), approximately the time that high levels of agnoprotein became detectable under our infection conditions. Thereafter, VP1 localized rapidly to the perinuclear region and to the nucleus. In contrast, in agnoprotein-minus mutant-infected CV-1P cells, perinuclear-nuclear accumulation of VP1 occurred much less efficiently; a significantly greater fraction of cells with predominantly cytoplasmic fluorescence was observed up to 48 h p.i. At 48 and 60 h p.i., more cells with largely perinuclear and little nuclear staining were seen than in WT-infected controls. In similar analyses with stably transfected cell lines constitutively expressing the agnoprotein, VP1 localized to the nucleus before 30 h p.i., regardless of the infecting virus. Delayed nuclear entry of VP1 in a mutant which makes no agnoprotein was also overcome in a revertant which has a second site point mutation in VP1. This suggests that an alteration of VP1 can partially overcome the defect of the agnogene mutation by enhancement of the rate of its own nuclear localization. Taken together, these results indicate that at least one function of the agnoprotein is to enhance the efficiency of perinuclear-nuclear localization of VP1.