We have used the ELISPOT method employing plastic ELISA plates without substrate in agar for the detection of single cells producing interferon-gamma (IFN-gamma) and interleukin-4 (IL-4). When using PBMC directly stimulated in the assay wells with T cell mitogens it was possible to measure production of human IFN-gamma at an earlier time point and with a higher sensitivity compared to conventional nitrocellulose plates. The plastic surface was not autostimulatory for IFN-gamma production, as seems to be the case for nitrocellulose surfaces. Compared to the use of nitrocellulose plates, the use of plastic ELISA plates is considerably cheaper and easier to perform. The increased sensitivity for cytokine detection, together with minimal autostimulatory properties of the detection surface, makes this method suitable for the detection of spontaneous low grade cytokine production from cells obtained in vivo.