The cuticle is a unique invisible oviduct secretion that protects avian eggs from bacterial penetration through gas exchange pores. Despite its importance, experimental evidence is lacking for where, when and what is responsible for its deposition. By using knowledge about the ovulatory cycle and oviposition we have manipulated cuticle deposition to obtain evidence on these key points. Cuticle deposition was measured using staining and spectrophotometry. Experimental evidence supports the location of cuticle deposition to be the shell gland pouch (uterus), not the vagina, and the time of deposition to be within the final hour before oviposition. Oviposition induced by arginine vasotocin or prostaglandin, the penultimate and ultimate factors for the induction of oviposition, produces an egg with no cuticle; therefore these factors are not responsible for cuticle secretion. Conversely, oviposition induced by GNRH, which mimics the normal events of ovulation and oviposition, results in a normal cuticle. There is no evidence that cuticle deposition differs at the end of a clutch and, therefore, there is no evidence that the ovulatory surge of progesterone affects cuticle deposition. Overall the results demonstrate that the cuticle is a specific secretion and is not merely an extension of the organic matrix of the shell. Cuticle deposition was found to be reduced by an environmental stressor and there is no co-dependence of the deposition of pigment and cuticle. Defining the basic facts surrounding cuticle deposition will help reduce contamination of hen's eggs and increase understanding of the strategies birds use to protect their eggs.