This study describes a serum-free medium in which adult rat islet beta-cells can be cultured in suspension for at least 9 days without a detectable loss in cell number or function. The medium is composed of Ham's F-10 with 10 mM glucose, 1% BSA, and 50 microM isobutylmethylxanthine. After 9 days of culture, beta-cell aggregates had preserved their initial DNA content, with more than 80% ultrastructurally intact cells. Their rates of glucose-inducible insulin synthesis (64 +/- 13 fmol/10(3) cells.2 h) and release (173 +/- 44 fmol/10(3) cells.2 h) were comparable to those previously determined in overnight cultured beta-cells. Their secretory response to 20 mM glucose plus 10(-8) M glucagon was biphasic and 10-fold elevated above the basal level. Their secretory and biosynthetic activities at basal (1.25 mM) glucose levels were significantly higher than after culture with serum. These elevated basal activities are attributed to a rise in the proportion of beta-cells with high content in pale secretory granules. Supplementing the serum-free medium with GH (1 micrograms/ml) plus glucagon (10(-8) M) further increased basal activities, leading to cellular degranulation and reduced hormone release after stimulation. Control cultures in Ham's F-10 with 10 mM glucose and 10% fetal calf serum reduced the initial DNA content by 40% and, consequently, the total amount of hormone synthesis and release. Surviving cells exhibited a lower secretory responsiveness than those recovered from serum-free medium; their lower basal activities coincided with an absence of cells with high content in pale granules. It is concluded that preservation of glucose-responsive beta-cells during suspension culture requires conditions that keep the cells recruited into glucose-dependent functions. Such a condition is achieved by the presently defined serum-free medium. It is characterized by the presence of a subpopulation of beta-cells with a high proportion of pale secretory granules.