Recombinant Chinese hamster ovary (CHO) cells expressing a high-level of chimeric antibody against S surface antigen of hepatitis B virus were obtained by co-transfection of heavy and light chain cDNA expression vectors into dihydrofolate reductase (dhfr)-deficient CHO cells and subsequent gene amplification in medium containing stepwise increments in methotrexate (MTX) level such as 0.02, 0.08, 0.32, 1.0, and 4.0 mu M. The highest producer (HP) subclone was isolated from each MTX level and was characterized with respect to cell growth and antibody production in the corresponding level of MTX. The specific growth rate of the HP subclone was inversely proportional to the MTX level. On the other hand, its specific antibody productivity (q(Ab)) rapidly increased with increasing MTX level up to 0.08 mu M, and thereafter, it gradually increased to 20 mu g/10(6) cells/day at 4 mu M MTX. Southern blot analysis showed that the enhanced q(Ab) at higher MTX level resulted from immunoglobulin (Ig) gene amplification. The stability of the HP subclones isolated at 0.02, 0.08, 0.32, and 1.0 mu M MTX in regard to antibody production was investigated during long-term culture in the absence of MTX. The q(Ab) of all subclones significantly decreased during the culture. However, the relative extent of decrease in q(Ab) was variable among the subclones. The HP subclone isolated at 1 mu M MTX was most stable and could retain 59% of the initial q(Ab) after 80 days of cultivation. Southern blot analysis showed that this decrease in q(Ab) of the subclones resulted mainly from the loss of Ig gene copies during long-term culture. Despite the decreased q(Ab), the HP subclone isolated at 1 mu M MTX could maintain high volumetric antibody productivity over three months because of improved cell growth rate during long-term culture. (C) 1998 John Wiley & Sons, Inc.