In order to achieve more stable and better yield, we conducted rejuvenation pruning of two main branches per walnut tree in spring 2015. 20-year-old trees of lateral fruiting varieties 'Lara', 'Fernor' and 'Fernette' were included into a study, and response to pruning was observed. Lenght of main branches, their apical and basal diameter, angle and number of nodes were measured before and after pruning. The same parameters were observed on two unpruned main branches per tree. In May, July and September we measured light interception of the canopy. At the end of 2015, we measured the length, basal and apical diameter and angle of newly formed annual shoots (AS). In the spring of 2016 we counted nodes, vegetative and fruting buds, and the number of female flowers and fruits on the AS. All varieties increased the number of the AS after a strong pruning, the most at the variety 'Fernette' (3.4 fold) and least at the variety 'Lara' (2.4 fold). The annual shoots on the pruned branches were 5.4 times longer and 1.1 times thicker at the variety 'Lara', 9.7 times longer and 1.3 times thicker at the variety 'Fernor' and 17 times longer and 1.8 times thicker at the veriety 'Fernette' than on the unpruned branches. There, the lenght of the AS as well as the number of nodes were approximately equal in all varieties. The AS on the pruned branches had more vegetative buds for 2.1 ('Fernette'), for 2.5 ('Lara') and for 3.8 ('Fernor') than the unpruned branches. At the veriety 'Fernette' the AS on the prunned branch showed approximatelly 4 fold more fuiting buds, female flowers, leaves and nuts than the unprunned branch. Measurements of photosynthetically active radiation showed that the light did not have such a strong effect on growth and characteristics of annual shoots. The effect of rejuvenation pruning was more dependent on the variety.