The effectiveness of the hok/sok plasmid stability locus and mechanism of cloned-gene loss was evaluated in shake-flask cultures. Addition of the hok/sok locus dramatically increased apparent plasmid segregational stability relative to the hok/sok(-) control. In terms of the number of generations before 10% of the population became plasmid-free, segregational stability was increased by 11- to 20-fold in different media in the absence of induction of the cloned-gene (hok/sok(+) plasmid stable for over 200 generations in all media tested). With constant expression of beta-galactosidase in the absence of antibiotic, the segregational stability of the plasmid containing hok/sok was increased more than 17- to 30-fold when beta-galactosidase was expressed at 7-15 wt % of total cell protein. Although the hok/sok system stabilized the plasmid well in four different media (Luria-Bertani (LB), LB glucose, M9C Trp, and a representative fed-batch medium), the ability of hok/sok to maintain the plasmid with induction of the cloned gene decreased as the complexity of the media increased. This result is better interpreted in terms of the influence of cloned-gene expression on plasmid maintenance; plasmid segregational stability decreased linearly as specific beta-galactosidase activity increased.