Introduction: Renal cell carcinoma (RCC) has a poor prognosis when metastasized to distant sites, although immunotherapy may offer a prolongation of survival in selected patient groups. Unfortunately, no treatment options remain when immunotherapy fails. In this phase IIa trial the tolerability and efficacy of the antifolate drug methotrexate-human serum albumin (MTX-HSA) were evaluated in patients with metastatic RCC who progressed after first-line immunotherapy. Patients and methods: A total of 17 patients started treatment, and 14 (12 men, 2 women) were evaluable for response according to the phase IIa Gehan design. Patients had prior tumor nephrectomy, were in relatively good general condition, had no impairment of renal, liver or bone marrow function, and had progressive metastatic disease after treatment with interferon-α(IFN-α) with or without cis-retinoic acid (EORTC protocols 30951 and 30947). MTX-HSA was given once a week intravenously on an outpatient basis at a dose of 50 mg/m2. The treatment interval was prolonged in those patients who had not yet recovered from previous toxicities. Results: Toxicity was manageable, relatively mild to moderate and reversible in most cases. Grade 2/3 mucositis (10/17) and grade 3 elevated transaminase levels (4/17) were most frequent, and in only one patient was a grade 4 thrombocytopenia reported. Of three inevaluable patients, one discontinued treatment due to drug-related toxicities. The mean administration interval was 12.1 days, and 7 of 14 evaluable patients had treatment intervals of 1 or 2 weeks. No objective responses were seen, although eight patients had stable disease (stabilization > 2 months) for up to 8 months (median 121 days). Conclusion: MTX-HSA was generally well tolerated and can be given on an outpatient basis, but no objective responses were seen in patients with metastatic RCC who had progressed after previous immunotherapy.