The ability to use magnetic nanoparticles for cell tracking, or for the delivery of nanoparticle-based therapeutic agents, requires a detailed understanding of probe metabolism and transport. Here we report on the development and metabolism of a dual fluorochrome version of our tat-CLIO nanoparticle termed Tat(FITC)-Cy3.5-CLIO. The nanoparticle features an FITC label on the tat peptide and a Cy3.5 dye directly attached to the cross-linked coating of dextran. This nanoparticle was rapidly internalized by HeLa cells, labeling 100% of cells in 45 min, with the amount of label per cell increasing linearly with time up to 3 h. Cells loaded with nanoparticles for 1 h retained 40-60% of their FITC and Cy3.5 labels over a period of 72 h in label-free media. Over a period of 144 h, or approximately 3.5 cell divisions, the T2 spin-spin relaxation time of cells was not significantly changed, indicating retention of the iron oxide among the dividing cell population. Using confocal microscopy and unfixed cells, both dyes were nuclear and perinuclear (broadly cytoplasmic) after Tat(FITC)-Cy3.5-CLIO labeling. Implications of the rapid labeling and slow excretion of the Tat(FITC)-Cy3.5-CLIO nanoparticle are discussed for cell tracking and drug delivery applications.