Rheum ribes L. is a perennial wild species. Young shoots and flower bunches are freshly consumed, and root and rhizomes are generally used for medicinal purposes. The aim of the present study was to improve the callus proliferation protocol for R. ribes L. under in vitro conditions. For callus induction, hypocotyl explants taken from 14-day old plantlets germinated in Murashige and Skoog (MS) media were cultured in MS media with 9 plant growth regulator (PGR) combinations containing 6-benzylaminopurine (BAP) (2, 3, and 4 mg/L) + naphthylacetic acid (NAA) (0.1, 0.5, and 1 mg/L). Then, for callus proliferation, 4 PGR combinations con-taining NAA (0.2 mg/L) + thidiazuron (TDZ) (0.5, 1, 2, and 3 mg) were used in the first set of experiments, and 36 PGR combinations containing BAP (1, 2, 3, and 4 mg/L) + indole-3-butyric acid (IBA) (0.2, 0.5, and 1 mg/L), BAP (1, 2, 3, and 4 mg/L) + NAA (0.2, 0.5, and 1 mg/L), and TDZ (1, 2, 3, and 4 mg/L) + NAA (0.2, 0.5, and 1 mg/L) were used in the second set of experiments. At the end of the second set of experiments, the greatest callus regeneration ratios were obtained due to the combinations including BAP and IBA as well as the low-dose TDZ-(especially 1 mg/L) and NAA-(0.2, 0.5, 1 mg/L) combinations. Regarding callus fresh weights, TDZ + NAA combinations were found to be more successful. The greatest callus fresh weight (12.7 +/- 0.4 g) was obtained from MS medium supplemented with 2 mg/L TDZ and 0.2 mg/L NAA.