Four structural analogs of benzoporphyrin derivative (BPD) have been studied and compared for photosensitizing activity in vitro. All analogs have an identical reduced tetrapyrrol porphyrin ring, and differ by the position of a cyclohexadiene ring (fused at either ring A or ring B of the porphyrin) and the presence of either two acid groups or one acid and one ester group at rings C and D of the porphyrin. Photosensitizer activity was tested with the M1 tumor cell line using an assay (the MTT assay) which detects mitochondrial hydrogenases as a measure of cell viability. This assay was shown to be equivalent to the standard clonogenicity or [3H]thymidine uptake assay. Comparative studies with the BPD analogs showed that the monoacid derivatives had equivalent cytotoxicity and were about five-fold more active than the diacid forms. This was the case whether the assays were performed in the presence or absence of fetal calf serum.