Parathyroid hormone (PTH) functions in part by regulating osteoblast cytokine expression. We recently demonstrated that PTH induced a rapid and transient increase in interleukin-6 (IL-6) mRNA expression in rat bones in vivo. To determine the molecular basis of this effect, we analyzed the human IL-6 promoter fused (-1,179 to +9) with the chloramphenicol acetyltransferase (CAT) reporter gene in stable transfections into human osteoblast-like osteosarcoma SaOS-2 cells. We compared the effects of PTH on IL-6 expression with adenylate cyclase activator forskolin, PKC activator phorbol 12-myristate 13-acetate (PMA), calcium ionophore A23187, interleukin-1 alpha (IL-1 alpha), prostaglandin E-2 (PGE-2), RS-66271 (a parathyroid hormone-related peptide analog), and platelet-derived growth factor-BB (PDGF-BB). Analyses of cell clones showed that IL-6 promoter expression was extremely low in the unstimulated state. Exposure to PTH (0.001-100 nM) for 12 h stimulated CAT expression in a dose-dependent manner (200-500% of control). Treatment with IL-1 alpha was more potent than PTH in inducing transcription of the IL-6 promoter (900-1,000%). Activation of the cAMP-PKA pathway by treatment with forskolin induced a comparable level of induction with PTH. Together, the effects of PTH and forskolin were additive. RS-66271, previously shown to have PTH-like effects, induced a comparable level of IL-6 promoter expression. When examined together, PTH+RS-66271 effects were comparable to PTH effects alone. Exposure to PGE-2, PMA, PDGF-BB, or A23187 for 12 h did not significantly alter IL-6 promoter expression. These results demonstrate PTH, forskolin, the PTHrP analog RS-66271, and IL-1 alpha stimulate IL-6 expression by stimulating gene transcription. The response to forskolin suggests that the messenger system mediated by PKA is sufficient to induce IL-6 expression.