Purpose: The purpose of this study was to evaluate penetration of self-etching primer to human sound dentin using confocal laser scanning microscope (CLSM) with fluorescent dye. Methods: The fluorescent dye Rhodamine B was mixed into self-etching primer of Clearfil Mega Bond FA (Kuraray Noritake Dental) at the concentration of 0, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1%, or 0.2%. All dentin surfaces were treated with Clearfil Mega Bond FA. Resin composites (Clearfil AP-X) were built up incrementally according to the manufacturer's instruction. 64 specimens were subjected to the observation of the interface of resin and dentin with CLSM and SEM. The 32 specimens were then serially sectioned into multiple sliced parts and then micro-tensile bond strength (MTBS) was measured in a universal testing machine. Results: Penetration of monomer into dentin via dentinal tubules was observed as moderate in the group of 0.07, 0.08, 0.09% and 0.1%. On the other hand, in the group of 0.2%, the color of Rhodamine B was too dark to identify the details of the dentinal tubules. There was no significant difference in the MTBS between the group of 0.05, 0.06 or 0.07% and control (p>0.05). The MTBS produced by the group 0.08, 0.09, 0.1% or 0.2% were significantly different from control (p<0.05). Conclusion: This study suggested that the application of CLSM was effective to evaluate penetration of self-etching primer to dentin. A high concentration of Rhodamine B influenced the bond strength. It was suggested that the appropriate concentration of Rhodamine B was 0.07% in the Mega Bond FA Primer.