Fibroblast migration is directed by gradients of platelet-derived growth factor (PDGF) during wound healing. As in other chemotactic systems, it has been shown recently that localized stimulation of intracellular phosphoinositide (PI) 3-kinase activity and production of 3′ PI lipids in the plasma membrane are important events in the signaling of spatially biased motility processes. In turn, 3′ PI localization depends on the effective diffusion coefficient, D, and turnover rate constant, k, of these lipids. Here we present a systematic and direct comparison of mathematical model calculations and experimental measurements to estimate the values of the effective 3′ PI diffusion coefficient, D, turnover rate constant, k, and other parameters in individual fibroblasts stimulated uniformly with PDGF. In the context of our uniform stimulation model, the values of D and k in each cell were typically estimated within 10-20% or less, and the mean values across all of the cells analyzed were D = 0.37 ± 0.25 μm2/s and k = 1.18 ± 0.54 min-1. In addition, we report that 3′ PI turnover is not affected by PDGF receptor signaling in our cells, allowing us to focus our attention on the regulation of 3′ PI production as this system is studied further.