The aims of the present study were to 1) examine the validity and relability of the laser probe, 2) to determine suitable cutoff points for identification of carious lesions and 3) to determine the significance of interference of exogenous stain in pits and fissures. One hundred and fifty nine predefined pit and fissure spots of 52 newly extracted teeth were examined 3 times by 2 examiners. The teeth were sectioned longitudinally right next ot the examination spots and examined in stereomicroscope to define the depth of carious lesions. The intraexaminer reliability was excellent (r=0.9) whereas the inter-examiner reliability was fair (r=0.7). The validity of the laser probe diagnosis on occlusal caries was fairly high, at the cutoff point of 11, the sensitivity and specificity for diagnosis of enamel caries were 0.76 and 0.77, respectively. The ability of the laser probe to distinguish between enamel and dentin caries was fair at the cutoff point of 35; the sensitivity and specificity were 0.57 and 0.76, respectively. However, stains retained deep in intact fissures significantly increased the laser probe score whereas the specificity of enamel caries identification decreased from 0.77 to 0.65. In conclusion, the laser probe appeared to be a reliable device for screening of non-cavitated occlusal caries in vitro. The specificity of the device was affected by stains within fissures.