This study was designed to investigate the relationship between the etched depth, penetration of sealants and marginal seal. Sixty unerupted lower first premolars extracted from patients for orthodontic reasons were thoroughly cleaned and stored in an antiseptic Ringer's solution at 4 degrees C. A "window" on the occlusal of 15 of the premolars, including both mesial and distal pits, was developed by painting nail varnish around the border of the occlusal surfaces. Etching was then done with 35% phosphoric acid for 60 seconds to all 60 premolars. Five of the "window" teeth were evaluated by microradiography to determine the etched depth of the superficial and subsurface enamel. Five were prepared for SEM analysis to observe the change of the superficial etched enamel surface. Another five "window" teeth were embedded in epoxy resin and sectioned parallel to the long axis of the tooth through the fissures in order to observe the subsurface depth of the etch by SEM analysis. The other 45 teeth were divided into three groups of 15 teeth each. Fissures of each group of teeth were sealed using Prisma-Shield (LD Caulk), Concise White Sealant (3M Dental Products) or Teethmate A (Kuraray) sealants and stored in water (37 degrees C) for 24 hours. They were then sectioned and demineralized before being examined by a scanning electron microscope. Photographs of secondary electron image (SEI) were done to gradate the resin-infiltrated enamel and resin tags for these sealants. After SEM observation, the 15 samples of each applied sealant were polished to a high gloss again and placed in a silver nitrate solution for 24 hours before being examined under the SEM equipped with a back-scatter electron detector. Data were then analyzed using the Welch and Student t-tests. Results showed that fissured enamel of unerupted human lower first premolars became porous after etching with 35% phosphoric acid. The low viscosity sealant Teethmate A (approximately 260 mPa.s), penetrated fully and formed a resin-infiltrated layer in enamel beyond the etched depth. However, the high viscosity sealants (Prisma-Shield and Concise White Sealant) did not penetrate enough to ensure that the acid-etched enamel was infiltrated sufficiently by the sealant to insure good marginal seals.