alpha 2-Macroglobulin (A2M) is a broad spectrum plasma protease inhibitor previously described in uterine effluents and recently demonstrated to bind and possibly modulate the functions of cytokines. As cytokines, proteases, and protease inhibitors are important in implantation and endometrial physiology, we sought to investigate and characterize the pattern of production of A2M in the endometrium. Endometrial tissues from different phases of the menstrual cycle were analyzed for A2M production. Tissues were incubated in methionine-free Minimum Essential Medium with [35S]methionine for 12 h at 37 C in 5% CO2. Conditioned media were immunoprecipitated with a polyclonal antibody to human A2M. Recovered proteins were resolved under reducing and nonreducing conditions by 5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by autoradiography. Immunohistochemistry was performed on both cryostat sections of OCT-embedded tissue and formalin-fixed paraffin-embedded tissue. The intensity of staining was evaluated, and a histochemical score was assigned. Comparisons between histochemical scores were performed using the nonparametric Wilcoxon rank sum test. Immunoprecipitation with A2M antibody yielded a single 320-kilodalton protein band under nonreducing conditions and a single 182-kilodalton band under reducing conditions. Both physical and immunological properties of the recovered protein were consistent with A2M. A2M was identified in all endometrial samples and represented approximately 2% of the total radiolabeled proteins produced. Immunohistochemical analysis revealed prominent stromal, but no glandular, staining for A2M throughout the menstrual cycle. The stromal staining in secretory endometrial samples was significantly more intense than that in proliferative samples. In the proliferative phase, staining was more intense in the spongiosum and basalis layers, and less intense in the superficial compactum layer. In the secretory phase, it remained very intense in the spongiosum, but less so in the basalis layer. These findings indicate that A2M is predominantly produced by the stromal component of endometrial tissue. This production is menstrual cycle dependent, with zonal differences in A2M expression within the endometrium, which may indicate a functional significance relevant to the process of implantation that merits further investigation.