Despite their important biological activity, lysosomes have been generally neglected as important primary targets of photosensitizers, because they are not easily accessible for experiments. This paper reviews factors favoring the localization of photosensitizers in lysosomes and the various experimental approaches which have been used so far for the characterization of the lysosomal staining by various photosensitizing dyes, including porphyrins, chlorins and phenoxazines. The experimental difficulties observed in combining several in vitro techniques for the unambiguous demonstration of lysosomal targeting are examined. New data on tetraphenylporphine derivatives and a pyropheophorbide, as well as previous data on photofrin II, are presented to illustrate the advantages and possibilities of microspectrofluorometry in the study of photosensitizer localization in single living cells. Both spectral and topographic information available from areas smaller than 1 microns2 make it possible to characterize fairly specific sites of localization through the use of specific and vital fluorescent probes of lysosomes, such as Lucifer Yellow. It is also shown by microspectrofluorometry on single living cells that the chronology of the photosensitized reactions induced by specific or unspecific lysosomal photosensitizers can be easily followed. The photosensitized lipofuscin formation observed at the plasma membrane level with the lysosomotropic tetraphenylporphine supports the contention that it is very rare to find a truly specific lysosomal photosensitizer.