A hybridoma secreting monoclonal antibody (mAb) specific to CD4 protein was generated. This monoclonal antibody, named MT4, was proved to be specific to CD4 protein as it reacted with CD4-DNA transfected COS cells, CD4+ cell lines and CD4+ lymphocytes. Furthermore, MT4 mAb inhibited the binding of standard CD4 monoclonal antibodies to CD4 proteins on CD4+ cells. To develop a home made reagent for CD4+ lymphocyte determination by flow cytometry, fluorescein isothiocyanate (FITC) was conjugated to MT4 mAb. To evaluate the developed reagent, 30 HIV infected and 30 healthy individuals were determined for CD4+ lymphocytes by using both a commercial Simultest reagent kit and home made FITC labeled MT4 mAb simultaneously. The study has shown that both percentages and absolute CD4+ lymphocyte counts obtained from both reagents were equivalent. The correlation coefficient for regression analysis was 0.995 and 0.996 for percentages and absolute CD4+ lymphocyte counts, respectively. The results suggest that home made FITC labeled MT4 reagent is an acceptable alternative reagent for monitoring CD4+ lymphocytes in blood samples by flow cytometry.