Thymosin beta(4) (Tbeta(4)), its sulfoxide, and thymosin beta(10 )(Tbeta(10)) were detected in human saliva and identified by different strategies based on RP HPLC coupled to electrospray multidimensional IT MS. Tbeta(4 )was almost always detected in whole saliva, its sulfoxide sporadically, Tbeta(10) rarely. Tbeta(4) was undetectable in parotid saliva and less concentrated in submandibular/sublingual saliva than in whole saliva. Analysis of gingival crevicular fluid revealed high relative amounts of Tbeta(4), Tbeta(4) sulfoxide, and Tbeta(10) in all the samples. Tbeta(4) mean concentration was 200 times higher in crevicular fluid (20 micromol/L, N = 9) than in whole saliva (0.1 micromol/L, N = 9). Crevicular fluid concentration of Tbeta(4 )(ca. 5% represented by its sulfoxide) and beta(10 )significantly correlated (r = 0.856; N = 9), and their ratio was about 5. A significant correlation was also observed between Tbeta(4 )concentrations in whole saliva and gingival crevicular fluid (r = 0.738; N = 9). Immunohistochemical analysis of the major salivary glands showed that immunoreactivity for Tbeta(4) is restricted to ductal cells, with minor degree of focal positivity in some acinar cells. On the whole, results indicate that gingival sulcus is a main, although not the sole, source for oral Tbeta(4 )and Tbeta(10).