As part of a program aimed at studying the feasibility of amide derivatives of methotrexate (MTX) as lipophilic prodrugs, with the aims of increasing passive cellular uptake and obtaining prolonged-release agents, we describe the synthesis of five long-chain alkyl bis(amides) of MTX, from decyl- to octadecylamide, by direct transamidation to the MTX diethyl ester. Compounds were subjected to a preliminary biological screening, to assess their inhibitory activity against bovine liver dihydrofolate reductase (DHFR) and in vitro antitumor activity against human leukemia CCRF-CEM cells. As a general trend, an increase in lipophilicity led to a linear reduction of enzyme inhibition; however, the bis(decyl)amide derivative showed a good intrinsic affinity for DHFR (IC50 6.41 nM), comparable to that of MTX diethyl ester and close to that of MTX (IC50 2.90 nM). In the antitumor assay, lower homologues (C10-C14) displayed an interesting activity profile, suggesting the desirability of additional studies with these and similar compounds.