A cationic, high-water-content hydrogel membrane composed of poly(vinyl alcohol) (PVA) and poly(ally-biguanido-co-allylamine) hydrochloride (PAB) with positively charged biguanido groups that resemble arginine residues was developed. The PAB was prepared by reacting poly(allylamine) hydrochloride (PAA) with guanyl-O-methyl isourea. PAB/PVA hydrogel membranes were prepared by repeated freezing and thawing. For comparison, hydrogel membranes composed of PAA and PVA were also prepared. The interaction between these hydrogel membranes and mouse fibroblast (L929) was studied by a cell culture method. The PAB hydrogel blend had a relatively low percentage of initial cell attachment. The cell growth on the PAB hydrogel membranes showed a maximum at 5 mol % PAB content that was as high as commercially available plastic films. However, cells on hydrogel membranes with 50 mol % PAB content and 0 mol % PAB content (only PVA) did not seem to grow; neither did the 5/95 PAA/PVA membranes. Water contact angles of hydrogel membranes did not vary with the PAB content. Morphology of the cell attachment was observed by SEM. On the PAB blend hydrogel surfaces, cells were not spindle-shaped and monolayers, but rather cells aggregated in spherical clusters.