The use of near-infrared (NIR) spectroscopy to interrogate deeper tissue volume has shown enormous potential for molecular-based non-invasive imaging when coupled with appropriate excitable dyes. As most of the breast cancers are hormone dependent hence determination of the hormonal receptor status gains paramount importance when deciding the treatment regime for the patient. Since proliferations of the breast cancer cells are often driven by estrogen, we focus on to developing a technique to detect estrogen receptor status. As a first step, the objective of this work was to synthesize and characterize one such novel NIR fluorescent (NIRF) conjugate, which could potentially be used to detect estrogen receptors. The conjugate was synthesized by ester formation between 17-b estradiol and a cyanine dye namely: bis-1, 1-(4-sulfobutyl) indotricarbocyanine-5-carboxylic acid, sodium salt. The cyanine dye is a hydrophilic derivative of indocyanine green (ICG). The ester formed was found to have an extra binding ability with the receptor cites as compared to ICG, which was established by the partition coefficient studies. This cyanine dye has a partition coefficient less than 0.005 as compared to that of ICG (>200). In addition the ester showed enhanced fluorescent quantum yield than ICG. The replacement of the sodium ion in the ester by a larger glucosammonium ion was found to enhance the hydrophilicity and reduce the toxic effect on the cell lines. The excitation and emission peaks for the conjugate were recorded in the NIR region as 750nm and 788nm respectively. The ester developed was tested on the breast cancer cell lines MCF-7 and found non-toxic. The tagging characteristics were pivotal determinants underlying the ability of the fluorescent conjugate in binding the estrogen receptor of the breast cancer cells. This technique offers the potential of non-invasive detection of hormone receptor status in vivo and may help in decreasing the load of unnecessary biopsies. Here, we have reported the progress made in the development of a novel NIR external contrast agent and the work is in progress to use this conjugate for the molecular-based, diagnostic imaging of breast cancer.© (2005) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.