Multi-colour imaging of immunofluorescently labelled tissue using confocal microscopy was accomplished by using colour addition theory. This new technique includes several improvements for immunolabelling: (1) the co-localization of two or more markers on one cell for the identification of specific cell populations; (2) the co-localization of two fluorescent dyes from secondary reagents for the identification of the cells; (3) a multi-step staining protocol with two primary antibodies originating from the same host species or with two or three biotin-conjugated primary antibodies. After image acquisition, colour segmentation/unmixing are applied to the single multi-colour image to generate multi-pseudo-channels for individual or co-localized fluorescent dyes. With this new technique, we have been able to visualize six cell populations simultaneously in the mouse lymph node and intestine. The efficiency of this method has also been demonstrated in the three-dimensional reconstruction of thick sections from mouse ileum. Our method is simple, efficient, and may be indispensable in experimental cell and tissue studies requiring multiple immunolabelling.