Magnetic-fluorescent nanocomposites enable the capabilities of both magnetic resonance imaging (MRI) and fluorescence-based technologies, such as confocal microscopy, to be exploited in single experiments(1). Here we report the in vitro evaluation of a novel Rhodamine B functionalised polyelectrolyte stabilised magnetic-fluorescent nanocomposite. Mixed glial cultures from neonatal mouse cortices were prepared, following decapitation in accordance with local ethical guidelines. Following mixed glia incubation in the presence of nanocomposite (5 μM) for 2 hours, nanocomposite uptake was assessed using confocal fluorescence microscopy and light microscopy. Live cell imaging data was acquired to investigate the method of nanocomposite internalization. Cell viability was measured in a mixed glia sample using the MTT assay, following incubation with nanocomposites (1, 5, 10, 20 μM) for 2, 18, 24, and 48 hours. The concentration of pro-inflammatory cytokines, IL-1β and IL-6, of mixed glia following a 2 hour incubation in the presence of the nanocomposites (1, 5, 10, 20 μM) was assessed using ELISA. MRI phantoms containing varying numbers of labelled cells in 0.5% Agarose were prepared. T2 and T2* relaxation times of these phantoms were calculated from images taken on a 7T Bruker spectrometer using multi-spin-echo (TE=2.09-59.09ms) and multi-gradient-echo (TE=10-200ms) sequences respectively. Tranmission electron microscopy (TEM) and confocal microscopy were used to assess nanocomposite behaviour following placement in a magnetic field. Confocal and light microscopy confirmed the internalization of the nanocomposites. Live cell imaging data suggests both phagocytosis and endocytosis as methods of nanocomposite internalization. No reduction was seen in cell viability. There was no increase in the concentrations of IL-1β or IL-6 following a 2 hour incubation. The contribution to MR contrast was calculated using γΔB ∝ (1/T2*) – (1/T2) and was linear (Pearson Correlation, R = 0.9967, p<0.001). TEM and confocal microscopy demonstrated the tendancy of the nanocomposites to form linear chain-like assemblies following placement in a magnetic field. The results confirm the potential use of these novel nanocomposites as bimodal contrast agents.