We have developed a new two-photon system for in vivo flow cytometry, thereby allowing us to simultaneously quantify different circulating populations in a single animal. The instrument was able to resolve minute-by-minute depletion dynamics of injected fluorescent microspheres at finer time scales than conventional flow cytometry. Also observed were the circulation dynamics of human MCF-7 and MDA-MB-435 breast cancer cells, which have low and high metastatic potential, respectively. After co-injection of both cell types into mice, markedly greater numbers of MCF-7 cells were present in the circulation at early time points. While low metastatic MCF-7 cells were cleared from the vascular system within 24 hours, detectable numbers of metastatic MDA-MB- 435 cells in the circulation remained constant over time. When we replace the commercial (80-MHz) NIR excitation laser with a reduced-repetition-rate (20-MHz) mode-locked oscillator, the signal is enhanced four-fold, enabling superior detection in blood of cell lines expressing fluorescent proteins tdTomato and mPlum (crosslabeled with DiI and DiD). Detection sensitivity versus incident laser power is understood in terms of detected event photon count distribution, which can be predicted with simple fluorophore distribution assumptions. The technique of two-color, two-photon flow cytometry greatly enhances the capabilities of ex vivo flow cytometry to investigate dynamics of circulating cells in cancer and other important diseases.© (2007) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.