Abstract Introduction: Chemotherapeutics typically distribute poorly into healthy brain due to the restrictive characteristics of the blood-brain barrier (BBB). Brain tissue immediately surrounding tumors (brain adjacent to tumor, BAT) is an important target for chemotherapy as tumorigenic cells proliferate and invade this region during tumor progression. Drug permeability in BAT is not well defined and discrepancies exist among studies estimating drug accumulation in this region. This study seeks to address these discrepancies by evaluating BAT permeability using quantitative fluorescent microscopy (QFM) in a preclinical model of brain metastases of breast cancer. Methods: A human breast cancer (MDA-MB-231Br) cell line transfected to stably express eGFP was injected into the peripheral circulation via the left cardiac ventricle and CNS metastases were allowed to grow for 4-6 weeks. After neurological symptoms developed, a fluorescence marker (Texas Red 625Da) was injected intravenously and allowed to circulate. Blood and brain concentrations of Texas Red were determined by QFM and regional brain uptake was determined. Lastly, we mapped fluorescent tracer concentration at 8 μm increments extending 300 μm inwardly and outwardly from the edge of selected metastases. Results: The permeability of Texas Red in MDA-MB-231Br brain metastases are heterogeneous, ranging nearly ∼10-fold between metastases for Texas Red 625Da. Quantitatively, most metastases (∼75%) exhibit modest increases in permeability (∼2 to 5-fold, p<0.05, n = 133), while ∼15% of metastases have greater than a 5-fold increase in permeability (p0.05, n = 15). Texas Red concentrations in BAT regions are elevated (p<0.05) relative to healthy brain and approximate healthy brain levels ∼100μm beyond tumor margins. The estimated accumulation of fluorescent tracer in BAT region reaches ∼30-40% of average concentrations observed in the core region of metastases. Conclusions: The BAT of experimental metastases of breast cancer exhibit elevated permeability relative to healthy brain. The increased accumulation of tracer in the BAT may result from either an increase in permeability at the local BAT region or from diffusion and flow of the tracer from the tumor core region into the BAT, or a combination thereof. However, this model suggests that this region accumulates approximately ∼50-60% less tracer than the respective tumor. Clinically, these data suggest that chemotherapeutic uptake in the BAT, where tumor cell replication and invasion occurs, remains limited and likely contributes to poor chemotherapeutic efficacy. Citation Format: Chris E. Adkins, Rajendar K. Mittapalli, Paul Lockman. Quantitative fluorescent microscopy to measure permeability in brain adjacent to tumor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3693. doi:10.1158/1538-7445.AM2015-3693