In the draft recommendations for production and control of OPV the WHO proposed new control methods: (i) mutant analysis with PCR and restriction enzyme cleavage (MAPREC) assay that allows evaluation of poliovirus population heterogeneity at the molecular level; (ii) neurovirulence (NV) test using transgenic mice susceptible to polioviruses and (iii) control of the seed lots for the presence of the simian virus 40 (SV40) DNA sequence. This paper is focused on our experience in the practical implementation of the new methods at the Institute of Poliomyelitis and Viral Encephalitides (IPVE). Using methods based on PCR we have demonstrated that working seed viruses used by IPVE for OPV production are free from SV40 DNA sequences. Our experience on the conduction of the OPV type 3 control using TgPVR21 mice NV test (seven vaccine lots) and the MAPREC assay (more than 150 samples of single harvests and monovalent bulks) showed that these methods may be used instead of the monkey NV test, because they could not pass the vaccine failed monkey NV test. The necessity for single harvests control is discussed.