The permeability barrier posed by cell membranes represents a challenge for the delivery of hydrophilic molecules into cells. We previously proposed that poly(2- alkylacrylic acid)s are endocytosed by cells into acidified vesicles and are there triggered by low pH to disrupt membranes and release the contents of endosomes/lysosomes to the cytosol. If this hypothesis is correct, these polymers could be valuable in drug delivery applications. This report provides functional comparisons of a family of three poly(2-alkylacrylic acid)s. Poly(2-propylacrylic acid) (PPAA), poly(2-ethylacrylic acid) (PEAA), and poly(2-methylacrylic acid) (PMAA) were compared in red blood cell haemolysis assays and in a lipoplex gene transfection assay. We also directly examined the ability of these polymers to disrupt endosomes and lysosomes in cultured human cells. Our results show that (i) unlike membrane-disruptive peptides, the endosomal-disruptive ability of poly(2-alkylacrylic acid)s cannot necessarily be predicted from their hemolytic activity at low pH, (ii) PPAA (but not PEAA or PMAA) potently facilitates gene transfection by cationic lipoplexes, and (iii) endocytosed poly(2-alkylacrylic acid)s are triggered by lumenal acidification to selectively disrupt endosomes (not lysosomes) and release their contents to the cytosol. These results will facilitate the rational design of future endosomal-disrupting polymers for drug delivery.