The aim of this study was to evaluate the marginal integrity of four self-etching adhesives (Prompt L-Pop III, Resulcin Aqua Prime & MonoBond N, AC Bond, AC Bond + Desensitizer) and three total-etch adhesives (OptiBond FL, Excite, Gluma Comfort Bond) in two different application protocols. Standardized MOD cavities (n = 40) were prepared in 280 extracted human teeth (one cervical margin in cementum, one in enamel). In one application protocol, the adhesive was applied prior to placement of the Tofflemire matrix (n = 20); in the other, the metal matrix was placed first (n = 20). Light curing was carried out from the occlusal aspect. After thermocycling (5000 times, 5°C and 55°C), specimens were immersed in 2% methylene blue for 10 s. Then the median percentages of dye-penetrated margins (surface analysis) were determined separately for the cervical cementum, cervical enamel, and lateral enamel margins of the proximal boxes. At the cervical cementum margins, statistically significantly worse results were obtained for all adhesives when the matrix system was placed prior to application of the adhesive. In the cervical enamel margins, OptiBond FL, Gluma Comfort Bond, and Prompt L-Pop III performed significantly better when the adhesive was applied prior to the matrix. On lateral enamel margins, only OptiBond FL and Resulcin Aqua Prime showed significantly less dye penetration if the matrix was placed first. Most of the total-etch materials produced marginal integrity significantly superior to that of the majority of the self-etching materials. Due to a significant influence of the application protocol on marginal integrity, it is strongly recommended to apply the adhesive system before placement of the matrix. Keywords: self-etching adhesives, Class II cavities, in vitro study, marginal integrity, dye penetration