Aiming to further our understanding of T cell-mediated suppression, we investigate the plausibility of the hypothesis that regulatory T cells suppress other T cells (target cells), while both cells are conjugated with one APC. We use a mathematical model to analyze the proliferation inhibition scored during in vitro suppression assays. This model is a radical simplification of cell culture reality, assuming that thymidine incorporation is proportional to the number of target cells that would instantaneously form conjugates with APCs that are free of regulatory cells. According to this model the inhibition index should be mainly determined by the number of regulatory cells per APC and should be insensitive to the number of target cells. We reanalyzed several published data sets, confirming this expectation. Furthermore, we demonstrate that the instantaneous inhibition index has an absolute limit as a function of the number of regulatory cells per APC. By calculating this limit we find that the model can explain the data under two non-mutually exclusive conditions. First, only approximately 15% of APCs used in the suppression assays form conjugates with T cells. Second, the growth of the regulatory cell population depends on the target cells, such that the number of regulatory cells per APC increases when they are cocultured with target cells and overcomes its limit. However, if neither of these testable conditions is fulfilled, then one could conclude that suppression in vitro does not require the formation of multicellular conjugates.