In primary cultures of rat hepatocytes, 16,16-dimethylprostaglandin E2 (16,16-dimethyl PGE2), a biologically active analogue of prostaglandin E2 (PGE2), stimulated the basal rate of [14C]glucose incorporation into glycogen. 16,16-Dimethyl PGE2 caused concentration-dependent stimulation (ED50: 10(-8) M) with a maximum 2-3 h after its addition. Prostaglandin E1 (PGE1), PGE2 and prostaglandin F2 alpha (PGF2 alpha) stimulated also the incorporation, but less effectively than 16,16-dimethyl PGE2. However, prostaglandin D2 (PGD2) did not show such effect. Cellular glycogen analysis revealed that PGE2 and 16,16-dimethyl PGE2 increased a net glycogen accumulation time-dependently. Pretreatment of the cultured hepatocytes with pertussis toxin blocked the effects of PGE2 and 16,16-dimethyl PGE2 completely and concentration-dependently. These findings indicate that E-series prostaglandins have significant effects on hepatic glycogenesis via pertussis-toxin-sensitive G protein, in addition to their inhibitory effects on hormone-stimulated glycogenolysis reported previously (Okumura, T., Sago, T. and Saito, K. (1988) Prostaglandins 36, 463-475).