In the course of our studies on compartment-specific lipid-mediated cell regulation, we identified an intimate connection between ceramides (Cers) and the mitochondria-dependent death-signaling pathways. Here, we report on a new class of cationic Cer mimics, dubbed ceramidoids, designed to act as organelle-targeted sphingolipids (SPLs), based on conjugates of Cer and dihydroceramide (dhCer) with pyridinium salts (CCPS and dhCCPS, respectively). Ceramidoids having the pyridinium salt unit (PSU) placed internally (alpha and gamma- CCPS) or as a tether (omega-CCPS) in the N-acyl moiety were prepared by N-acylation of sphingoid bases with different omega-bromo acids or pyridine carboxylic acid chlorides following capping with respective pyridines or alkyl bromides. Consistent with their design, these analogs, showed a significantly improved solubility in water, well-resolved NMR spectra in D(2)O, broadly modified hydrophobicity, fast cellular uptake, and higher anticancer activities in cells in comparison to uncharged counterparts. Structure-activity relationship (SAR) studies in MCF-7 breast carcinoma cells revealed that the location of the PSU and its overall chain length affected markedly the cytotoxic effects of these ceramidoids. All omega-CCPSs were more potent (IC(50/48 h): 0.6-8.0 microM) than their alpha/gamma-CCPS (IC(50/48 h): 8-20 microM) or D-erythro-C6-Cer (IC(50/48 h): 15 microM) analogs. omega-DhCCPSs were also moderately potent (IC(50/48 h): 2.5-12.5 microM). Long-chain omega-dhCCPSs were rapidly and efficiently oxidized in cells to the corresponding omega-CCPSs, as established by LC-MS analysis. CCPS analogs also induced acute changes in the levels and composition of endogenous Cers (upregulation of C16-, C14-, and C18-Cers, and downregulation of C24:0- and C24:1-Cers). These novel ceramidoids illustrate the feasibility of compartment-targeted lipids, and they should be useful in cell-based studies as well as potential novel therapeutics.