Picornavirus infection induces the proliferation and rearrangement of intracellular membranes in response to the synthesis of nonstructural proteins, including 3A. We have previously shown that changes in 3A are associated with the inability of a Taiwanese strain of foot-and-mouth disease virus (FMDV) (OTai) to grow in bovine cells and cause disease in cattle, although the virus grows to high titers in porcine cells and is highly virulent in pigs (C. W. Beard and P. W. Mason, 2000, J. Virol. 74, 987-991). To study if differences in the distribution of 3A could account for the species specificity of OTai, we compared the localization of the OTai 3A with a bovine-virulent 3A (serotype A12) in keratinocytes prepared from the tongues of cattle and pigs. Following either infection of keratinocytes or transfection with 3A we were unable to discern differences in 3A distribution in either species of keratinocyte, independent of the strain of virus (or 3A) utilized. In both cell types, 3A distributed in a pattern that overlapped with an endoplasmic reticulum (ER) marker protein, calreticulin (CRT). Furthermore, although FMDV infection or transfection with 3A did not result in a gross redistribution of CRT, both virus infection and 3A transfection disrupted the Golgi. Other picornaviruses that disrupt Golgi function are sensitive to brefeldin A (BFA), a fungal metabolite that interferes with retrograde transport between the Golgi and the ER. Interestingly, BFA has little effect on FMDV replication, suggesting that FMDV may acquire cellular membranes into its replication complexes in a manner different from that of other picornaviruses.