Expression of integrin αvβ3 is upregulated in a number of cancers including colon, pancreas, lung and breast. Previous studies demonstrated that near infrared (NIR) fluorescent probes designed to target αvβ3 accumulated both in vitro and in vivo in αvβ3-positive tumor cells. To evaluate the selectivity of some NIR-labeled RGD peptides for αvβ3, the molecular probes were incubated in different cells, including the αvβ3-positive U87 and A549 cells, and αvβ3-negative HT29 cells. Whereas the RGD compounds tested internalized in the A549 cells, their uptake by the HT29 cell line, which is positive for αvβ5 and αvβ6, was low. The uptake of these probes in U87 depended on the structural features of the compounds. Further studies with functional blocking antibodies showed that the internalization in the αvβ3-positive cells may be mediated by different integrin receptor subtypes. The preliminary results suggest that the internalization of linear RGD peptides is mediated by the αvβ3 heterodimer but rearrangement of the peptide sequence could alter the selectivity of the molecular probes for different integrin subunits in the dimeric α and β proteins. Thus, a careful choice of RGD peptides can be used to monitor the functional status of different integrins in cells and tissues.