Culture of polioviruses from clinical samples is the gold-standard method for virological surveillance in the world-wide initiative to eradicate wild-type polioviruses. Two poliovirus-sensitive cell lines of human origin were used originally by the laboratories of the World Health Organisation (WHO) global poliovirus network. However, the cell lines used, Hep2 and RD, also support cytopathic growth of a variety of non-poliovirus enteroviruses. This can make detection of polioviruses in samples with mixtures of viruses difficult and time consuming. The development of mouse cell lines that express the gene for the human cellular receptor for polioviruses allows selective poliovirus culture, because very few non-poliovirus enteroviruses grow in these murine cells. A WHO Collaborative Study was initiated to test one such cell line, L20B, and to compare under routine conditions the sensitivity and selectivity of L20B cells against RD and Hep2 cells. Five laboratories in countries endemic or recently endemic for wild polioviruses participated. A total of 425 samples were tested prospectively in all three cell lines and there was a clear and consistent trend for greater sensitivity for polioviruses in L20B cells. Overall, 148/160 polioviruses were detected in L20B cells compared with 89/160 in RD and 98/ 160 in Hep2. In part, this finding was due to detection in L20B cells of polioviruses from samples that also contained non-poliovirus enteroviruses in which the poliovirus was masked in RD or Hep2 cells. However, L20B cells were also significantly more sensitive for poliovirus than either RD or Hep2 cells in three of the five study laboratories. The L20B cells were completely selective for polioviruses, as 0/89 wild type non-poliovirus enteroviruses produced cytopathic effect in L20B cells. Finally, L20B cells provided a diagnosis of poliovirus infection in the same time as RD and Hep2 cells from samples that contained poliovirus only, but substantially more quickly for samples that contained another enterovirus. Taken together, these data indicate that L20B cells simplify primary diagnosis of poliovirus from clinical samples and as a result they have been introduced for routine use by laboratories of the WHO global poliovirus network.