We here report improved synthesis and in vitro interactions of amphiphilic hydrogel nanoparticles with the macrophage cell line J774A.1. Nanoparticles comprising dispersed hydrophobic nanodomains of poly(propylene glycol) within a continuous phase of hydrophilic poly (ethylene glycol) (PEG) were prepared via inverse emulsion crosslinking polymerization, using acrylated PEG and Pluronic" F127 as macromonomer blocks. Functionality and fluorescent labeling were achieved through incorporation of reactive comonomers and a posteriori reaction with fluorescein, respectively. When introduced to a static cell culture of adhered J774A.1 macrophages, the cells internalized these hydrogel nanoparticles in a dose- and time-dependent manner through clathrin-mediated and other pathways. Amphiphilic nanoparticle uptake was however dramatically lower than that of a model system (Fluospheres (R)) and similar to PEG-coated colloids reported in the literature, which are considered "stealth." Our findings support the potential of the nanoparticles presented here as long-circulating drug carriers. (C) 2009 Wiley Periodicals, Inc. J Biomed Mater Res 93A: 1557-1565, 2010