Introduction: Primary cell culture is obtained by isolation of cells directly from the tissue. By cultivation under appropriate conditions, primary cells grow and proliferate, but have a limited life span. The basic steps in establishing a primary culture cell include obtaining a sample, tissue isolation, dissection or tissue degradation and cultivation in culture vessels. Objective: The aim of the study is to establish the primary culture of mononuclear mouse cells and to determine the conditions necessary for their growth. Materials and methods: The blood from three female CB7BL/6N strains was used in the study. The mice were raised at the vivarium of Faculty of Medicine in Osijek. Lymphocytes were isolated from the blood by the density gradient method using a Lymphoprep solution. Cultivated cells are stored in a CO_2 incubator (Shel lab) under a 5% CO_2 atmosphere and a temperature of 37 ° C. An RPMI 1640 defined medium was used to maintain the primary culture of mononuclear cells. The number of viable cells was determined in a viability test with Trypan blue color in the Bürker-Türk chamber. Results: Primary culture of peripheral mononuclear cells was established. The number of viable cells in culture on the first day was 213,75 x 〖10〗^4 cells/ ml. Conclusion: The establishment of primary culture of peripheral mononuclear cells of a mouse is possible by adhering to the rules of aseptic technique and cultivation under strictly controlled conditions in which cells can survive up to eight days.