This study evaluated Knoop microhardness (KHN) and dental plaque composition of human enamel irradiated with Nd:YAG laser and subjected to in situ cariogenic challenge. Although in vitro studies have shown that Nd:YAG laser irradiation results in a reduction of enamel mineral loss after acid challenge, this hypothesis has not been tested using an in situ caries model. Acrylic devices containing lased and non-lased human enamel slabs were used for 28 days by eight subjects highly colonized by S. mutans. Devices were immersed in a sucrose solution (20%) eight times daily in order to better simulate the cariogenic challenge. After the experimental period, no differences in dental plaque composition were observed between lased and non-lased enamel slabs indicating that all slabs were exposed to equivalent cariogenic challenges. Cross sections of enamel slabs were then measured for Knoop microhardness at 10, 20, 40, 60, 80, 120, and 180 microm in depth from the outer exposed surface. Un-lased enamel slabs not subjected to the in situ cariogenic challenge were also used as control. Enamel slabs from the two in situ groups exhibited lower hardness when compared to control. Lased enamel showed higher hardness values up to 60 microm in depth than the un-lased enamel exposed to in situ cariogenic challenge. The results suggest that the irradiation of Nd:YAG laser of human enamel appears to be effective for increasing enamel hardness and thus decreasing the effect of acid attack.