In-nest observations of the solitary bee, Megachile rotundata (F.), revealed that nesting females apply olfactory cues to nests for nest recognition. On their way in and out of the nest, females drag the abdomen along the entire length of the nest, and sometimes deposit fluid droplets from the tip of the abdomen. The removal of bee-marked sections of the nest resulted in hesitation and searching behavior by females, indicating the loss of olfactory cues used for nest recognition. Chemical analysis of female cuticles and the deposits inside marked nesting tubes revealed the presence of hydrocarbons, wax esters, fatty aldehydes, and fatty alcohol acetate esters. Chemical compositions were similar across tube samples, but proportionally different from cuticular extracts. These findings reveal the importance of lipids as chemical signals for nest recognition and suggest that the nest-marking cues are derived from a source in addition to, or other than, the female cuticle.