The objective of this research project is to investigate the biocompatibility of N-isopropylacrylamide (NIPAAm) monomers and poly(N-isopropylacrylamide) (PNIPAAm) nanoparticles in vitro. PNIPAAm nanoparticles of different sizes were synthesized and characterized by transmission electron microscopy and dynamic light scattering. Cytotoxicity studies using MTS assays were conducted on fibroblasts, smooth muscle cells, and endothelial cells. In addition, the concentration of NIPAAm monomers remaining on PNIPAAm nanoparticles was determined using bromination and spectrophotometry. The cytotoxicity results did not show a significant difference in cell survival when cells were exposed to different particle sizes (100, 300, and 500 nm). Dose studies showed that all three cell types exposed to 100 nm PNIPAAm nanoparticles at concentrations less than or equal to 5 mg/mL were compatible, while cells exposed to NIPAAm monomers exhibited toxicity even at very low concentrations. We also found that 1 mg/mL concentration of 100 nm PNIPAAm nanoparticles was cytocompatible for 4 days, whereas NIPAAm monomers were cytotoxic after 24 h of exposure. Photomicrographs showed altered morphology in cells exposed to NIPAAm monomers, while cells exposed to PNIPAAm nanoparticles maintained their normal morphology. Finally, a very low concentration of NIPAAm monomers remained on the PNIPAAm nanoparticles after synthesis and dialysis. Our results demonstrate that NIPAAm monomers are cytotoxic, whereas PNIPAAm nanoparticles are compatible at 5 mg/mL concentration or below for fibrobasts, smooth muscle cells, and endothelial cells.