A procedure for rapid in vitro propagation of the rare, endangered, ornamental and medicinal plant Pulsatilla pratensis (L.) Miller ssp. nigricans (Störck) Zämelis (Ranunculaceae) is described. Sterilized seeds were germinated on media of different pH values containing full or half-strength Murashige and Skoog macronutrients (MS) or supplemented with GA₃ alone or in combination with BA. Germination percentage was highest (58.3%) on basal MS medium pH 5.8 augmented with 2.9 μM GA₃ while the greatest number of shoots were achieved on MS and half-strength MS media supplemented with both growth regulators regardless of pH value. For multiplication of Pulsatilla pratensis ssp. nigricans, the same media with slight modifications were tested. The highest multiplication rate was achieved on MS medium pH 5.8 containing 2.9 μM GA₃ and 0.5 μM BA. Shoots excised from multiple shoot cultures were rooted on MS and half-strength MS media containing three different auxins (IAA, IBA and NAA) at three concentrations. The percentage of rooted plantlets was highest (83%) on MS medium supplemented with 8.5 μM IAA.