We have carried out the adaptation of BHK-21 cells to two serum free (Ex Cell 520 and HyQ PF CHO) and three animal protein free media: Ex Cell 302, HyQ PF CHO MPS and Rencyte BHK. After a direct switch or a gradual adaptation, we have achieved BHK-21 cells growth in the following media: HyQ PF CHO, HyQ PF CHO MPS, Rencyte BHK and Ex Cell 302. The most suitable media for BHK-21 cells growth, with respect to cell density and specific growth rate, were HyQ PF CHO and HyQ PF CHO MPS. Hence we have selected these media to study cell growth and the production of rabies virus. Kinetic studies of cell growth in spinner flasks using the selected media have shown that a maximal cell density of 2x10(6) cells x ml(-1) was reached in both media. For rabies virus production, the viral titer obtained was 1.7x10(6) FFU x ml(-1) in HyQ PF CHO as well as in HyQ PF CHO MPS medium. The optimization of rabies virus production by BHK-21 cells grown in a 2 l bioreactor using the selected media, pointed to the following parameters: culture mode, perfusion rate and multiplicity of infection (MOI), as being the critical factors for achieving a good virus yield. When tested in mice, the activity of the experimental vaccines prepared on HyQ PF CHO MPS medium has shown a protective activity that meets WHO requirements.