Films of agarose gel impregnated with polylysine spotted from an aqueous solution have been characterized by atomic force microscopy performed in deionized water. An increase in contact stiffness of the composite substrate on the spotted areas has been observed, for increasing polylysine concentration. For the considered agarose layer thickness of approximately 0.9 microm when dry, the polylysine concentration threshold for stiffening is as low as approximately 0.1 mg/mL. Above this threshold the stiffening coefficient increases slightly with concentration in the considered range (up to 0.7 mg/mL), reaching a highest value of approximately 2.3. For concentrations >or=0.3 mg/mL the stiffening at the polylysine spots was also accompanied by a locally lower film thickness. For accurate quantification of the stiffness, representative force-distance curves extracted from the respective regions of interest (spots and agarose substrate off the spots) have been processed. The Hertz model of purely elastic tip-surface interaction has been adopted, with appropriate hypothesis on both tip shape and optimum indentation depth. The resulting Young's moduli of the agarose layer and of the polylysine spots are approximately 45 kPa and approximately 95 kPa, respectively, with an estimated uncertainty of approximately 15%.