Recent studies suggest that low molecular weight (LMW) GTP-binding proteins are involved in the vesicular transport of proteins along the secretory pathway. In order to determine whether LMW GTP-binding proteins are potentially involved in the regulated secretion of atrial natriuretic factor (ANF) from the atrial myocyte, rat atrial secretory granules were purified and the LMW GTP-binding proteins associated with these granules were detected in [alpha-32P]GTP-blotting experiments. Three LMW GTP-binding proteins, displaying molecular weights of 23,000, 25,000 and 29,000, were usually observed in atrial secretory granule preparations. The 29 kDa GTP-binding protein (G29), however, was typically the predominantly labelled band. The binding of [alpha-32P]GTP to G29 was guanine nucleotide specific and magnesium-dependent. Trypsin treatment of intact secretory granules markedly reduced the binding of [alpha-32P]GTP to G29. G29 is tightly associated with the granule membrane, as evidenced by its resistance to solubilization by high ionic strength buffers but its sensitivity to solubilization with sodium cholate. When secretory granule preparations of rat anterior pituitary glands were examined for the presence of LMW GTP-binding proteins, a different pattern of LMW GTP-binding proteins was observed which did not include a predominantly labelled 29 kDa protein. The results of this study indicate that: (1) LMW GTP-binding proteins are present on atrial secretory granules and may therefore be involved in the regulated secretion of ANF and (2) tissue-specific differences exist between the LMW GTP-binding proteins associated with secretory granules.