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 dramitically increasedapparent plasmid segregational stability 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 β-galactosidase in the absence of an tibiotic, the segregational stability of the plasmid containing hok/sok was incresed more than 17- to 30-fold when β-galactosidase was expressed at 7–15 wt % of total cell protein. Although the hok/sok system stabilized the plasmid well infour different media (Luria-Bertani (LB), LB glucose, M9C Trp, and a representative fedbatch 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 plasmidmaintenance; plasmid segregational stability decreased linearly as specificβ-galactosidase activity increased. © 1994 John Wiley & Sons, Inc.