A characteristic feature of trisomy 16 mouse conceptuses is a failure of their eyelids to close. This defect was investigated by examining ocular development in serially sectioned heads of trisomy 16 and normal littermate fetuses from 10 to 18 gestational days. Other heads were examined by using scanning electron microscopy. Between 10 and 15 days, trisomy 16 ocular structures were delayed, but there was no striking abnormal morphology. At 16 days, when the eyelids were closed and fused in normal mice, trisomic eyes had a large cell mass near the inner canthus that protruded between the open lids. The mass was covered by bulbar conjunctiva and cells of the mass were continuous with developing corneal tissue. The mass was not present in the eyes of normal mice on any gestational day and was not present in trisomic eyes at 17 and 18 days, when the lids began to show varying degrees of closure. Based on its positioning at the inner canthus, the mass may represent a transient hyperplasia of the developing semilunar fold which physically impedes lid closure in the trisomic conceptuses. Previously, the defect has been attributed to the trisomy 16 conceptus's overall pattern of growth retardation and delayed development. Masses such as those seen in the trisomic eyes have not been observed in other murine lid-gap defects that have been investigated. A second finding in this study is that trisomic eyes are positioned more superiorly in the head than normal eyes. This variation may be related to alterations in cranial base morphology that are associated with trisomy 16.