Structural changes in poly(3-hydroxybutyrate) (P3HB) monofilaments (diameter similar to 90 mu m), induced by annealing under tensile stress (1 h with 1.6-40 MPa loading at 80-130 degrees C) have been investigated with synchrotron wide-angle x-ray diffraction (WAXD) and polarized attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. Melt-spun P3HB fibers consist of an amorphous phase, a crystalline alpha-phase and a mesophase. Specific IR bands in s-polarized (electric field vector along the fiber axis) spectra have been identified to arise from the mesophase. For fibers annealed at low stress (1.6 MPa), the mesophase content practically disappears but can be recovered with tensile drawing. In-situ WAXD and polarized ATR-FTIR studies, performed during cyclic tensile loading/unloading, have revealed that the mesophase formation/dissolution is highly reversible in fibers annealed at low stress. This reversibility behavior, and the lack of off-axis reflections in WAXD patterns, support the theory that the mesophase is made of conformationally disordered and stretched chains, which are mainly located in-between alpha-crystals.