A complete serum-free process without washing steps and medium exchange before infection for influenza A virus vaccine production (equine and human) is described for cultivation in roller bottles and in a 5-L stirred tank microcarrier system. Adherent Madin-Darby canine kidney cells (MDCK) were adapted from growth in serum containing GMEM medium to growth in serum-free Ex-Cell MDCK medium. Roller bottle experiments showed that the medium exchange step, typically required for serum containing vaccine production processes, could be omitted without losses in virus titre and without limitations in glucose or glutamine supply in the cultivation medium. The serum-free medium could even be used glutamine-free as it contained pyruvate, resulting in very low levels of ammonia. Cell attachment onto microcarriers was critical. Therefore, microcarriers had to be preconditioned in medium. Also, trypsin concentration used for inoculum preparation had to be reduced. After these modifications 1.3 x 10(6)cells/mL were obtained after 97 h (2g/L Cytodex 1) of cell growth. Maximum virus titres of 2.3-2.9 log HA units/100 microL were obtained from infections with a multiplicity of infection (moi) of 0.05-0.10 for human and equine influenza A virus. Metabolite and amino acid profiles as well as on-line data for the serum-free process are compared with the serum containing process. Omission of the medium exchange before infection clearly simplified the process and reduced sterility risks due to washing steps.