To assess the expression of rabies virus G-glycoprotein (RVGP) expression using Semliki Forest virus as a vector in combination with BHK-21 cells cultured in suspension. A multilevel factorial design was used to quantify effects of temperature (33-37 A degrees C), fresh medium addition after the viral adsorption step (100-200 % with respect to the initial cell suspension volume before infection) and harvest time (8-40 h) on RVGP production. Experimental runs were performed in 24-well cell culture plates at a multiplicity of infection (MOI) of 16. An additional experiment in spinner-flask was performed at MOI of 9, using the optimal conditions determined in cell culture plates. Values for temperature, fresh medium addition and harvest time of 33 A degrees C, 100 % and 16 h, respectively, ensured the optimal RVGP production in culture plates. The volumetric yield (239 ng ml(-1)) in these conditions was higher than that reported previously for adherent cell culture. In spinner-flasks, the volumetric yield was improved (559 ng ml(-1)). These results establish the basis for designing bioprocess to produce RVGP.