Piritrexim is a new antifolate that has shown activity in methotrexate-resistant tumors. Gemcitabine is an antimetabolite similar in structure to cytosine arabinoside with early studies demonstrating activity in a variety of cancers. It also has apparent synergistic activity with antifolates from initial work in tumor models. Paclitaxel is an antimicrotubule agent that has a wide spectrum of activity against a variety of solid tumors. The combination of gemcitabine, paclitaxel, and piritrexim was assessed in this phase I trial. Thirty patients were enrolled. The starting doses were piritrexim 25 mg orally twice daily (days 1-4, 15-18), paclitaxel 75 mg/m2 (days 1, 15), and gemcitabine 750 mg/m2 (days 1, 15), which then was escalated in a stepwise fashion. Four patients achieved stable disease while on study, whereas one patient with a poorly differentiated neuroendocrine tumor achieved a partial response. The main toxicity was myelosuppression. The maximum tolerated dose was thought to be piritrexim 25 mg orally three times daily (days 1-4), paclitaxel 150 to 175 mg/m2 (days 1, 15), and gemcitabine 1,000 mg/m2 (days 1, 15). The combination of these new antifolates with paclitaxel and gemcitabine appears safe and should be considered for phase II trials in known responsive tumors such as transitional cell carcinomas.