This research was conducted through a factorial experiment with two factors including culture media (DKW and WPM) and Benzyl Adenine (BA) at three concentrations (0: control, 0.5 and 1.0 µm) for establishment of single node explants of Persian walnut, selected genotype 305, based on a completely randomized design. Thus, the effect of two cytokinin hormones (BA and Kinetin) in three concentrations (4.4, 6.6 and 8.8 µm) with IBA in two concentrations (0.05 and 0.1 µm) were studied on the proliferation of explants established on the selected medium. The results obtained from the proliferation step were analyzed through unbalanced complete randomized design. The aim of the establishment test was to determine the most effective medium for transmission to the next step. The mean comparison of the data for number of nodes and leaves showed that DKW medium performed better than WPM. Also, no significant difference was observed between 0.5 and 1 µm of BA concentrations. But a significant difference was observed between treatments with 0.5 and 1 µm of BA concentrations. Based on these results, the DKW medium was continued to be used in the next experiments. Results of mean comparison of data showed that the higher rates of growth indices were observed with 8.8 µm BA which only had a significant difference with 4.4 µm of BA. Overall results at this stage showed that BA performed better than Kinetin, and the rate of all investigated traits increased when cytokinin concentration increased. All data were analyzed with SAS software and the means of data were compared using Duncan's multiple range test.