The diffusion coefficient of a small hydrophobic probe in poly(lactide-co-glycolide) (PLGA) microparticles was determined by laser scanning confocal microscopy (LSCM). PLGA microparticles preincubated in a physiological buffer for various times were immersed in an aqueous solution of the pH-insensitive fluorescent probe bodipy at 37 degreesC. Probe concentration gradients inside individual microparticle matrices were then recorded by LSCM, which were accurately fit by the solution to Fick's second law of diffusion to determine an effective probe diffusion coefficient (D) in the polymer matrix. Values of D varied less than expected from the blank eroding polymer and were in the range (3-10) x 10(-12) cm(2)/s. The apparent polymer-water partition coefficient of the probe was also determined to be roughly 20, indicative of strong partitioning into the polymer phase. The diffusion of bodipy in microparticles varied over 3 orders of magnitude between 22 and 43 degreesC, indicative of transport control in the polymer phase as opposed to a pore diffusion mechanism. The diffusion model also predicted exceptionally well the release of probe encapsulated in microparticles when a time-averaged D, which had been determined in blank microparticles by LSCM, was used. Values of D for bodipy in PLGA microparticles encapsulating bovine serum album were of similar value ((3-24) x 10(-12) cm(2)/s) but better reflected the multiphasic behavior characteristic of PLGA erosion. The LSCM method described here is simple, nondestructive, and accurate and can be used to study the diffusion inside a single polymer microparticle.