The effect of serum type and concentration on recombinant protein expression in vaccinia virus infected HeLa S3 cells was studied in both static and suspension culture. A model heterologous protein, β- galactosidase (β-gal), was used. Calf and horse sera in the range of 0.5- 10%(v/v) were investigated. In static culture, the calf serum concentration did not show any significant influence on the β-gal production which was almost completed within 24 h postinfection (pi). Higher horse serum concentration, on the other hand, resulted in higher β-gal concentration which continued to increase until 48 h pi. Total β-gal concentrations in 0.5% calf serum at 24 h pi and 10% horse serum at 48 h pi were 2.2 ± 0.7 and 2.2 ± 0.1 IU/ml, respectively. In suspension culture, both sera showed their respective effects on the β-gal production similar to those observed in static culture, indicating that the cultivation method had little influence on β-gal production. Accordingly, the use of 0.5% calf serum after virus infection is recommended for economical β-gal production.