Histatins constitute a distinct family of human salivary antimicrobial peptides, of which histatins 1, 3 and 5 are the most abundant. To evaluate salivary gland-specific differences in histatin secretion, we used the recently developed histatin-zinc precipitation method to quantify histatins and to assess daily variations in secretions. Stimulated pure secretions from parotid glands (HPS) and submandibular/sublingual glands (SMSL) were collected from 10 different subjects at four different times of the day (9:35 a.m.; 12:40 p.m.; 2:50 p.m. and 5:00 p.m.). Zinc precipitation and subsequent reversed phase HPLC analysis were performed to determine concentrations of histatins 1, 3 and 5 with reference to purified histatin standards. Both HPS and SMSL secretions displayed daily variations in histatin concentrations. HPS values showed a maximum at mid-day and SMSL samples showed a maximum in the morning. Mean daily histatin concentrations were almost three fold higher in SMSL than in HPS. Mean histatin 1, 3 and 5 concentrations in HPS from 10 subjects ranged from 0.7 to 2.8, 0.6 to 4.3 and 1.0 to 4.3mg%, respectively. The corresponding means in SMSL were 2.8-12.2, 1.5-7.5 and 2.6-9.0mg%, respectively. Remarkably, although histatins constitute only 3-10% of total protein in these secretions, an almost perfect correlation between total protein and total histatin concentrations was observed for both glands. Despite a broad range in histatin concentrations between individuals, this study demonstrated a hitherto unidentified daily variation in histatin concentrations in HPS and SMSL secretions and a differential expression pattern which might have functional implications.