Fluid within the uterine cavity provides the microenvironment for preimplantation blastocyst development and early implantation. Analysis of uterine fluid sampled by aspiration or lavage provides a view of this microenvironment but the similarity or otherwise of the sample components is not known. This study compared proteins in aspirates versus lavage samples taken sequentially from the same women, using surface-enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS), multiplex cytokine assays, and an activity assay for proprotein convertase 6. Both lavage and aspiration enabled analysis of uterine fluid components, but they provided substantially different protein profiles. Although there were many similarities in overall protein profiles and most specific proteins examined were detected in both fluids, these were neither qualitatively nor quantitatively comparable within each participant. A likely explanation is that lavage samples the entire uterine cavity including washing the endometrial surface (glycocalyx), whereas aspiration sampling is very local.