Spodoptera frugiperda (IPLB-SF-21) insect cells were grown in shake-flasks and infected with a temperature-sensitive baculovirus to express the gene of chloramphenicol acetyl transferase (CAT) in serum-free medium (SF-900) and two serum-supplemented media (IPL-41 and Grace's). In temperature-shift experiments (cell growth at 33 degrees C followed by virus replication at 27 degrees C 3-4 days later), virus and CAT production were much poorer in the serum-free medium than in serum-supplemented media, though cell growth was virtually the same in the different media tested. In all the three media, highest virus and CAT titers were obtained at the lowest MOI (multiplicity of infection 0.02). This result is contrary to that obtained in constant-temperature culture (27 degrees C for both cell growth and virus replication). Virus and CAT production was greatly improved when the entire culture was run at constant temperature. It appeared that infected cells were severely damaged at 33 degrees C (6 degrees C above the optimal 27 degrees C), resulting in little or no virus and protein production. As a result of these temperature-shift experiments, a larger-scale (14 1 air-lift bioreactor) serum-free culture of Sf-9 insect cells was conducted at constant temperature (27 degrees C) to produce recombinant protein (beta-galactosidase). A cell density as high as 1 x 10(7) cells.ml-1, and a beta-gal concentration of up to 104,000 unit.ml-1 were achieved.