Periodontal disease is characterised by proteolytic processes involving enzymes that are released by host immune cells and periodontal bacteria. These enzymes, when detectable in whole saliva, may serve as valuable diagnostic markers for disease states and progression. Because the substrate specificities of salivary proteases in periodontal health and disease are poorly characterised, we probed these activities using several relevant substrates: (i) gelatin and collagen type IV; (ii) the Arg/Lys–rich human salivary substrate histatin-5; and (iii) a histatin-derived synthetic analog benzyloxycarbonyl-Arg-Gly-Tyr-Arg-methyl cumaryl amide (Z-RGYR-MCA). Substrate degradation was assessed in gel (zymography) and in solution. Whole saliva supernatant enzyme activities directed at gelatin, quantified from the 42 kDa, 92 kDa and 130 kDa bands in the zymograms, were 1.3, 1.4 and 2.0-fold higher, respectively, in the periodontal patient group (P < 0.01), consistent with enhanced activities observed towards collagen type IV. On the other hand, histatin 5 degraded equally fast in healthy and periodontal patients' whole saliva supernatant samples (P > 0.10). Likewise, the hydrolysis rates of the Z-RGYR-MCA substrate were the same in the healthy and periodontal patient groups (P > 0.10). In conclusion, gelatinolytic/collagenolytic activities but not trypsin-like activities in human saliva differentiate health from periodontal disease and may thus provide an adjuvant to diagnosis for monitoring disease activity.