The substituents of drug molecules and the specific amino acid residues of P-glycoprotein (P-gp) implicated in drug/protein interactions are largely unknown. We have used a series of colchicine analogs modified on the A, B, and C rings to identify the discrete chemical groups on the colchicine molecule that are required for recognition by P-gp. For this, the toxicity of these analogs was tested on independent cell clones expressing either of the two mouse mdr genes, mdr1 and mdr3, known to confer multidrug resistance. Modifications of the methoxy groups on the A and C rings modulated cellular toxicity but had no effect on P-gp recognition; however, modifications at the C7 position of the B ring, in particular the removal of the nitrogen atom of the acetamido group, had a dramatic effect. Analogs bearing a hydrogen at that position were not substrates for P-gp. The importance of the nitrogen at C7 was independently verified in thiocolchicine and allocolchicine analogs similarly modified, although overall levels of resistance to these compounds were somewhat reduced compared to their colchicine counterparts. The study of allocolchicine congeners bearing a six-carbon C ring and of two other analogs completely lacking a B ring suggested that intact B and C rings were important for interaction with P-gp. These results suggest that the structural determinants for cytotoxicity (tubulin binding) and P-gp recognition map to nonoverlapping sites in the colchicine analogs analyzed. Examination of calculated molar refractivities (CMR) revealed that only compounds showing CMR values greater than 9.7 were P-gp substrates.(ABSTRACT TRUNCATED AT 250 WORDS)