Previous studies have shown that hypericin is an excellent diagnostic tool for the fluorescence detection of carcinoma in situ in the human bladder. The present work was performed to get a better insight into the mechanism of cellular uptake of hypericin (HYP) using RT-112 human papillary TCC cells of the bladder. Using lipophilic hypericin acid amide derivatives like hypericin acid hexylamide (AM6), hypericin acid octylamide (AM8) and hypericin acid dodecylamide (AM12), the effect of increased lipophilicity on the binding to serum proteins was investigated, as well as the cellular accumulation and permeation, both in 2-D and 3-D cell conditions. Density-gradient ultracentrifugation of the compounds pre-incubated with fetal bovine serum (FBS) showed that HYP and to a lesser extent AM6 predominantly bind to LDL, whereas AM8 and especially AM12 preferably associate with HDL. The cellular accumulation of the compounds did not significantly differ when LDL or AcLDL was supplemented to medium, and with all compounds the highest uptake could be observed in case of medium without supplements. Using medium without supplements it was further observed that the compounds with the highest lipophilicity accumulated substantially less in RT-112 cells. We further found a significant difference in the intracellular concentration of AM8 and AM12 when LDL or FBS was supplemented to MEM, but not in case of HYP and AM6. Of particular interest, AM8 and especially AM12 showed enhanced intraspheroidal permeation in the presence of FBS. It is believed that the relative stronger binding to HDL reduces the intracellular accumulation, as seen in the 2-D conditions, and therefore increases the probability of paracellular transport in a 3-D multicellular system by passive diffusion. In conclusion the data suggest that the amount of free hypericin or its lipophilic congener determines the extent of intracellular accumulation. This concentration is both determined and limited by binding to different lipoproteins present in the medium, and by the formation of stable homoaggregates. The findings further highlight AM8 and AM12 as compounds better tailored for paracellular transport than HYP itself and therefore as potentially very interesting diagnostic tools for TCC lesions in the bladder.