To determine the effect of hyperosmotic stress on the monoclonal antibody (MAb) production by calcium-alginate-immobilized S3H5/γ2bA2 hybridoma cells, the osmolalities of medium in the MAb production stage were varied through the addition of NaCl. The specific MAb productivity (q(MAb)) of immobilized cells exposed to abrupt hyperosmotic stress (398 mOsm/kg) was increased by 55% when compared with that of immobilized cells in the control culture (286 mOsm/kg). Furthermore, this enhancement of q(MAb) was not transient. Abrupt increase in osmolality, however, inhibited cell growth, resulting in no increase in volumetric MAb productivity (r(MAb)). On the other hand, gradual increase in osmolality allowed further cell growth while maintaining the enhanced q(MAb) of immobilized cells. The q(MAb) of immobilized cells at 395 mOsm/kg was 0.661 ± 0.019 μg/10 cells/h, which is almost identical to that of immobilized cells exposed to abrupt osmotic stress. Accordingly, the r(MAb) was increased by ca. 40% when compared with that in the control immobilized cell culture. This enhancement in r(MAb) of immobilized S3H5/γ2bA2 hybridoma cells by applying gradual osmotic stress suggests the potential of using hyperosmolar medium in other perfusion culture systems for improved MAb production.