A recombinant Autographa californica baculovirus expressing secreted alkaline phosphatase (SEAP) gene was used to evaluate the expression of a secreted glycoprotein in eight insect cell lines derived from Spodoptera frugiperda, Trichoplusia ni, Mamestra brassicae and Estigmene acrea. Because cell density was found to influence protein production, SEAP production was evaluated at optimal cell densities for each cell line on both a per cell and per milliliter basis. On a per cell basis, the T. ni-derived BTI-TN-5B1-4 cells produced a minimum of 20-fold more SEAP than the S. frugiperda-derived Sf9 or Sf2l cell lines and a minimum of 9-fold more than any of the other cell lines growing in serum-containing medium. On a per milliliter basis, BTI-TN-5B1-4 cells produced a minimum of fivefold more SEAP than any of the other cell lines tested. Using cell lines that were adapted to serum-free medium, SEAP yields were the same or better than their counterparts in serum-containing medium. At 3 days postinoculation, extracellular SEAP activity ranged from 59 to 85% of total SEAP activity with cell lines grown in serum-free and serum-containing media.