Rab GDP dissociation inhibitor (GDI) plays an important role in regulating the GDP/GTP cycle of small GTP binding proteins of the Rab family. It also regulates their association to membranes. The small family of Rab-GDI consists of several closely related isoforms, the functional differences between which are still unknown. Here we show that multiple GDI isoforms are expressed in rat parotid gland and that the individual GDI isoforms have a characteristic expression both at the RNA and at the protein level, during the parotid secretory cycle. GDI alpha, the major isoform in brain, is expressed throughout the secretory process and is equally distributed between cytoplasmic and membranous fractions. In contrast, an isoform related to, but different from GDI beta is found predominantly in the cytoplasmic fraction and its expression is detected only after beta-adrenergic stimulation of the gland, at the end of the secretion phase, when exocytosis is already completed. The induction of such a GDI isoform at the beginning of the recovery stage correlates with the expression pattern of Rab1 and Rab5, but not Rab2 and Rab4. Our results suggest different functional roles for multiple GDI isoforms along the secretion and recovery phases in rat parotid gland. (C) 1997 Academic Press.