Mesenchymal stem cells positive to surface antigen STRO-1 were isolated from regenerating antlers of red deer (Cervus elaphus) and fallow deer (Dama dama) using a magnetic-activated cell sorting (MACS) method. In this study we analysed factors potentially affecting the number of STRO-1(+) cells in the cell cultures. With regard to the STRO-1 antigen, we evaluated data from 188 MACS separation procedures of cell cultures cultivated in Dulbecco’s Modified Eagle Medium and 10% fetal calf serum of four fallow deer males (130 procedures) and four red deer males (58 procedures). The analysed factors were the sampling site of the antler or the pedicle, cell passage and type of the cell culture (mixed or STRO-1 negative cell cultures). The percentage of obtained STRO-1(+) cells varied greatly from 0.4 to 38.9% for fallow deer and from 1.8 to 16.5% for red deer. We have not found any significant influence of the sampling site. The passage and the type of culture were significant factors for both fallow and red deer cells. The highest numbers of STRO-1(+) cells were obtained from the second passage from both fallow and red deer cell cultures (24.6 and 5.5%, respectively). Our experiment revealed that we can maximise the number of STRO-1(+) cells in the cultures by manipulating the cultivation factors.