A callus induction and plant regeneration protocol was developed from leaf and petiole explants of the endemic Astragalus nezaketae. Explants were cultured on Murashige and Skoog medium (MS) supplemented with different plant growth regulators (PGRs) [α-naphthaleneacetic acid (NAA), benzyladenine (BA), 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin (Kin), thidiazuron (TDZ)]. The combinations and concentrations of PGRs were shown significant variations for the frequency of callus formation, appearence of callus and the potential of callus differentiation. NAA x BA have been found highly affective in callusing and plant regeneration. Other PGRs have not resulted in callus differentiation for shoot formation. The highest number of shoots (6/explants) was obtained from leaf explants cultured on MS with 0.5 mg/l NAA and 4 mg/l BA. The regenerated shoots transferred to rooting medium (MS with 0.5 mg/l indole-3-butyric acid) were successfully rooted (100%) and showed rapid elongation. Rooted plantlets were acclimatized in pots containing 1:1 mixture of peat and perlite. © 2010 by Pontificia Universidad Católica de Valparaíso, Chile.