We recently compared the efficiency of six picornaviral internal ribosome entry segments (IRESes) and the hepatitis C virus (HCV) IRES for their ability to drive internal initiation of translationin vitro. Here we present the results of a similar comparison performed in six different cultured cell lines infected with a recombinant vaccinia virus expressing the T7 polymerase and transfected with dicistronic plasmids. The IRESes could be divided into three groups: (i) the cardiovirus and aphthovirus IRESes (and the HCV element) direct internal initiation efficiently in all cell lines tested; (ii) the enterovirus and rhinovirus IRESes are at least equally efficient in several cell lines, but are extremely inefficient in certain cell types; and (iii) the hepatitis A virus IRES is incapable of directing efficient internal initiation in any of the cell lines used (including human hepatocytes). These are the same three groups found when IRESes were classified according to their activitiesin vitro, or according to sequence homologies. In a mouse neuronal cell line, the poliovirus and other type I IRESes were not functional in an artificial bicistronic context. However, infectious poliovirions were produced efficiently after transfection of these cells with a genomic length RNA. Furthermore, activity of the type I IRESes was dramatically increased upon co-expression of the poliovirus 2A proteinase, demonstrating that while IRES efficiency may vary considerably from one cell type to another, at least in some cases viral proteins are capable of overcoming cell-specific translational defects.