Abstract— The influence of cell heterogeneity on response to photodynamic treatment (PDT) has been investigated using the human colon adenocarcinoma line WiDr, grown as spheroids and exposed to hematoporphyrin derivative. The spheroids show a marked spheroid size-dependent resistance to PDT. Using a flow cytometer, cell sub-populations have been separated, on the basis of drug fluorescence, from single cell suspensions prepared from 500 μm diameter spheroids. Cells low in fluorescence have been shown to be resistant to PDT, have a smaller median cell volume, and be enhanced in G1-type cells. These cells also show reduced low density lipoprotein uptake. The results suggest that spheroid size-dependent resistance to PDT is related to a decreasing growth fraction with increasing spheroid size. Heterogeneity of drug uptake could be a potential limitation to clinical PDT.