Recently we have shown that post-exposure treatment with povidone iodine (PI) protects against nitrogen and sulfur mustard-induced skin lesions. Since proteolytic activity is involved in skin damage caused by chemical irritants, we have studied the effect of iodine on mechlorethamine (HN2)-induced skin collagenolytic activities in the haired guinea pig model. The matrix metalloproteinase-9 (MMP-9) activity increased by 30, 46, 12 and 23% after 3, 24, 48 and 72 h of HN2 exposure, respectively, whereas the MMP-2 was elevated by 8, 65, 8 and 30%, respectively. Topical treatment with PI at 15 and 120 min after HN2 exposure decreased the MMP-9 activity by 67% and 60%, respectively, when skin was analyzed 3 h after exposure. The same trend was observed in the MMP-2 and MMP-1 activities after PI treatment. A stronger effect of PI treatment 15 min following exposure was observed in skin analyzed 24 h after exposure, i.e. a decrease of 83% and 88% in MMP-9 and MMP-2 activities, respectively. Similar findings were observed with an interval of 120 min between HN2 exposure and PI treatment. A much weaker effect was observed on MMP-1 activity. A similar trend of PI-induced reduction in the three types of collagenase activity was found in skin analyzed 48 and 72 h after exposure. Reduced collagenolytic activity may serve as one of the mechanisms by which iodine protects the skin against chemical insult.