All viruses with a positive-stranded RNA genome replicate their genomic RNA in association with membranes from the host cell. Here we demonstrate a novel organelle source of replication membranes for human rhinovirus 1A (HRV-1A). HRV-1A infection induces fragmentation of the Golgi apparatus, and Golgi membranes are rearranged into vesicles of approximately 250-500 nm diameter. The newly distributed Golgi membranes co-localize with viral RNA replication templates, strongly suggesting that the observed vesicles are the sites of viral RNA replication. Expression of the HRV-1A 3A protein induces alterations in the Golgi staining pattern similar to those seen during viral infection, and expressed 3A localizes to the Golgi-derived membranes. Taken together, these data show that in HRV-1A infection, the 3A protein plays a role in fragmenting the Golgi complex and generating vesicles that are used as the site of viral RNA replication.