Estramustine, and derivatives of estramustine with a charged substituent at position 17 on the estrogen moiety, have been investigated for their effects on bovine brain microtubules in vitro. The negatively charged estramustine phosphate has been found previously to be a microtubule-associated protein (MAP)-dependent microtubule inhibitor [Wallin M, Deinum J and Fridén B, FEBS Lett 179: 289-293, 1985]. In the present study the binding of estramustine phosphate to MAP2 and tau was investigated. Both these MAPs were found to have two to three binding sites for estramustine phosphate which is compatible with the reported number of basic amino acid repeats of these MAPs, considered to be the ultimate tubulin binding domains. The Kd for the binding of estramustine phosphate to MAP2 was estimated to be 20 microM at 4 degrees, and for the binding of tau, 200 microM. The rate of dissociation was very low (T1/2 greater than 2 hr), which indicates that the binding of estramustine phosphate may stabilize the protein-drug complex by changing the protein conformation. Two new negatively charged estramustine derivatives, estramustine sulphate and estramustine glucuronide, were found to be similar MAP-dependent microtubule inhibitors. The concentration for 50% inhibition of assembly was 100 microM for the sulphate derivative, the same as found previously for estramustine phosphate, and 250 microM for the more bulky estramustine glucuronide. A positively charged derivative, estramustine sarcosinate, did not inhibit microtubule assembly or alter the composition of the coassembled MAPs. The morphology of the microtubules was, however, affected. The uncharged estramustine bound to both tubulin and MAPs, but no effects were seen on microtubule assembly, the composition of coassembled MAPs or the microtubule morphology. Our results suggest that only negatively charged estramustine derivatives have a MAP-dependent microtubule inhibitory effect. The two new negatively charged derivatives could therefore be valuable tools in the study of tubulin-MAP interactions. The results also confirm that these interactions between tubulin and MAPs are mainly electrostatic.