Valeriana officinalis, commonly known as valerian, is native to Europe and Asia and has economic importance as a medicinal plant. The effects of auxins (2,4-D and NAA) on callus induction were studied in vitro using basal piece of leaf + petiole (L1) and middle piece of leaf (L2) explants of V. officinalis. The best callus formation (95.83%) was reached on MS containing 2,4-D (1.5 mg/l!1) + KIN (1 mg/l!1) although there was no significant difference with 2,4-D (2 mg/l!1) + KIN (0.5 or 1 mg/l!1) on L2 explants. All calli successfully developed roots on callus induction media, a feature that can be used in the future for a large-scale production, saving time and cost. Among cytokinins (BAP, TDZ, and KIN) tested for callus differentiation, the highest regeneration (62.5%), shoot number (5.87) and shoot length (5.96 cm), occurred on a combination of BAP (0.5 mg/l!1) + IBA (0.5 mg/l!1). The results revealed that the effect of explant type on callogenesis was also significant. In contrast to KIN or TDZ, addition of IBA to BAP showed positive synergistic effect for plant regeneration and shoot number per callus.