Amphiphilic copolymers based on poly(2-hydroxyethyl aspartamide) (PHEA) formed self-aggregates for the entrapment and release of methotrexate (MTX) by physical entrapment and chemical conjugation. In physical entrapment, MTX was partitioned into hydrophobic domains in self-aggregates of PHEA grafted with octadecyl chains (PHEA-C18) and the amount of the entrapped drug increased linearly by 3.39 mg per the degree of substitution of grafted octadecyl groups. The amphiphilic nature of the drug induced a large initial release in the buffer medium, irrespective of the amount of octadecyl chains. However, PEG-grafted PHEA-C18 copolymers conjugated with MTX, ConG, formed a micelle-like structure by self-association of the conjugates and suppressed the initial large release. The alkyl grafting lowered the CAC, meaning enhancement of aqueous stability. The release was accelerated in pH 10.0 by rapid hydrolysis of ester linkage by base-catalyzed cleavage, while it was significantly reduced at pH 5.0.