Transduction of intracellular signals requires multiple protein-protein and cellular structure interactions. These interactions affect the mobility of the involved proteins; therefore mobility measurements could provide insight into these interactions. Fluorescence recovery after photobleaching (FRAP) is an effective tool to analyze intracellular protein mobility. In the present study we report the application of FRAP to monitor intracellular signaling by fibroblast growth factor receptor (FGFR1), a transmembrane and a nuclear protein. FGFs have been shown to stimulate phosphatidyl inositol 3-kinase (PI3K) activity through activation of a high affinity FGFR. The interaction of FGFR1 phosphotyrosines with the regulatory subunit, p85alpha, of PI3K has been indicated by FGFR1 and p85alpha co-immunoprecipitation in different cell types. It is important to monitor these interactions in live cells, as cell lysing can show false interactions. The speed of protein movement and the percent of mobile molecules in live cells could be used to monitor direct or indirect interactions between proteins, as well as the formation of protein complexes and their association with cellular structures. We have used FRAP to study the mobility of p85alpha in the cytoplasm and nucleus of live human TE671 cells and to determine whether the interaction with FGFR1 will influence p85alpha mobility. Using TE671 medulloblastoma cells as a model, we demonstrate that the mobility of p85alpha-fused to enhanced green fluorescent protein (EGFP) is significantly reduced when cotransfected with FGFR1, in both the cytoplasm and nucleus. This effect was abolished by deletion of the tyrosine kinase domain from FGFR I (the catalytic, signaling region of the receptor), even though it changed the mobile fraction of p85alpha-EGFP. We find FRAP to be a valuable technique for protein interaction studies and conclude that p85alpha mobility is regulated by cytoplasmic/nuclear FGFR1 in the cytoplasmic and nuclear compartments of live cells.