The membrane transport properties of a series of dialkyloxacarbocyanine [DiOCn(3)] dyes in multidrug-resistant KB cell lines were investigated to determine the influence of alkyl chain length on the ability of p-glycoprotein (i) to protect cells from the toxicity of the dyes and (ii) to affect the plasma membrane flux of the dyes. Cytotoxicity assays revealed that increased levels of p-glycoprotein led to increased resistance to the toxicity of the DiOCn(3) relative to the sensitive KB-3-1 parent line. This resistance could be fully or partially reversed by 10 microM verapamil. Monitoring of DiOCn(3) fluorescence changes allowed the measurement of accumulation and efflux rates for the dyes in the parent and two resistant cell lines at 1.5-s resolution. The flux of DiOCn(3) into and out of the KB85 and KBV1 cell lines was shown to be dramatically different from the parental KB-3-1 line when n < 5, while the transport properties of n = 7 were identical in the three cell lines examined. The membrane transport properties were shown not to be correlated with the 7-day toxicity of DiOCn(3). Verapamil affected the kinetic processes of DiOC2-5(3) involving redistribution of the dyes within the cells once they had initially passed the plasma membrane. Fluorescence microscopy was used to show no alteration in the subcellular distribution of the DiOCn(3), in response to neither chain length nor cell line. Our results indicate that an alkyl chain length of 5 carbons is the critical length necessary for p-glycoprotein to affect membrane transport of DiOCn(3) but not to protect the cells from the cytotoxicity of the dyes.