P‐glycoprotein (P‐gp), a product of the MDR1 gene, is an important factor in the turnover of many drugs and xenobiotics. Recent reports have suggested that P‐gp can also be involved in the transport of cytokines. The aim of this study was to examine the role of P‐gp in cytokine release from phytohaemagglutinin (PHA)‐stimulated peripheral blood mononuclear cells (MNCs) as well as in the release of cytokines from MNCs treated with methotrexate (MTX) and dexamethasone (DEX). The study was carried out on PHA‐stimulated MNC from 10 healthy subjects. Flow cytometry was applied to measure interleukin (IL)‐2, IL‐4, IL‐6, IL‐10, interferon (IFN)‐γ and tumour necrosis factor (TNF)‐α levels in the culture supernatants. In the experiments verapamil (VER) and P‐gp specific monoclonal antibodies (mAb) (clone 17F9) were used to inhibit P‐gp function. P‐gp inhibitors suppressed the release of IL‐2, IL‐4, IFN‐γ and TNF‐α from PHA‐stimulated MNC, whereas release of IL‐6 and IL‐10 remained unaffected. VER and mAb significantly decreased the release of IL‐2, IL‐4, TNF‐α and INF‐γ in MNC cultures treated with MTX or DEX. The results of this study suggest that P‐gp may be involved in the transmembrane transport of some cytokines. Moreover, it seems that blocking of P‐gp function may influence the release of some cytokines from MNCs, displaying an additive inhibitory effect to DEX and MTX.