Trichoplusia ni-derived BTI-Tn5B1-4 (Tn-5B1-4) cells were. grown in 50 mL spinner flasks in four different formulations of serum-free medium (SFM). The objectives of this work were to determine the capacity of this cell line to grow and support virus replication in suspension culture in different SFM. Maximum cell densities exceeding 3.0 x 10(6) cells/ml were attained for all cell/medium combinations. Likewise, cell doubling times were all within 20-22 h. Infection studies with a wild-type isolate of Autographa californica nuclear polyhedrosis virus (AcMNPV) yielded between 156 and 187 occlusion bodies (OBs)/cell. The lethal effect of AcMNPV-OBs produced in this cell line in the various types of media was demonstrated in vivo using T. ni neonate larvae. The percent mortality at a single dose (1.0 x 10(6) OBs/mL) ranged from 40 to 60% at 4 days post-inoculation (p.i.). These results compared favorably with mortality rates found for AcMNPV-OBs produced in vivo. DNA quantification analysis, comparing polyhedra-derived viral DNA concentrations from an equal number of cell culture-and larval-derived OBs, was also determined. The larval-derived OB DNA concentration averaged 0.514 mu g/10(8) OBs, whereas the cell culture-derived viral DNA concentrations ranged from 0.347 to 0.710 mu g/10(8) OBs. These results suggest that no significant differences exist in DNA content between larvae-and cell culture-derived samples. Expression of the recombinant protein, secreted alkaline phosphatase (SEAP), ranged from 2.7 to 5.5 IU/mL for each cell/medium combination, which was consistent with previous static culture results for this same cell line in serum-containing TNM-FH medium. Electron microscopic analysis of OBs produced in vivo or in vitro revealed no obvious differences in packaging or number of enveloped nucleocapsids per OB between samples. However, AcMNPV-OBs from infected larvae appeared physically smaller (1.28 +/- 0.164 mu m) than those produced in cell culture (1.72 +/- 0.120). The Tn-5B1-4 cell line is adaptable to suspension culture in serum-free medium and is suitable for further development in the production of viruses and recombinant proteins on an industrial scale.