The antifolate aminopterin (AMPT) was developed before methotrexate (MTX), but was not clinically established or generally used due its increased toxicity compared to MTX. Recently, we reported on the increased metabolism of albumin conjugates such as methotrexate–albumin (MTX–SA) in malignant tumors and the feasibility of using albumin as a carrier for drug targeting. Consequently, AMPT was covalently bound to serum albumin (AMPT–SA) at a 1:1 molar ratio. Biodistribution, tolerability and efficacy of this novel conjugate were studied in Walker-256 (W-256) carcinoma-bearing rats. As compared to native albumin, the same biodistribution and plasma clearance were found for AMPT–SA, which achieved 20.1% tumor uptake (estimated uptake per g tumor 6.4%) within 24 h after i.v. administration in rats. In a randomized study, AMPT–SA, repeatedly i.v. injected, was compared with low-molecular-weight AMPT. Depending on the molar concentration, the maximum tolerated dose (MTD) of AMPT covalently bound to SA was twice that of unbound AMPT (three repeated injections of 1.0 mg AMPT–SA/kg body weight versus three repeated injections of 0.5 mg AMPT/kg body weight;p=0.0006). Efficacy was studied at the level of the MTD and MTD/2, and demonstrated that AMPT–SA was significantly more active. At the MTD/2 in W-256 carinoma-bearing rats, AMPT–SA achieved a 100% volume reduction and an optimal volume reduction during treatment/control (T/C) of 8.3% compared to a 53% volume reduction of AMPT and a T/C of 16.5% (p=0.032). Tumor relapses were reduced and occurred later in the AMPT–SA group (two tumor recurrences for AMPT–SA versus seven for AMPT;p=0.05). In this comparative study, the AMPT–SA conjugate showed high antitumor activityin vivoand a favorable toxicity compared to low-molecular-weight AMPT. These effects are attributed to the albumin carrier which seems to be an effective tool for selective tumor drug targeting.