Studying skin immune cells under various pathophysiological conditions is vital for understanding the nature of cutaneous inflammatory responses. Available methods of isolating cells from the skin have relatively low yield or require in vitro culture. To increase the effective isolation of skin immune cells, we used collagenase P treatment. The number of T cells obtained ex vivo using this technique was dramatically greater than that obtained with conventional methods, without the need for long-term culture. The phenotype and function of isolated cells were comparable with those of cells isolated by EDTA treatment. Collagenase P-based methods will enhance the ability to investigate lymphoid cell function in both healthy and diseased skin.