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OBJECTIVE: To enhance the laboratory diagnosis of ectopic pregnancy by determining levels of hCG, progesterone, estradiol (E2), and alpha-fetoprotein (AFP). METHODS: Serum samples and medical records were retrospectively analyzed from 100 gynecologic patients for whom quantitative hCG determination had been ordered. Clinical data and levels of hCG, progesterone, E2, and AFP were examined by univariate and multivariate logistic analyses. RESULTS: Progesterone, hCG, and E2 were highest in viable pregnancies, whereas AFP tended to be higher in ectopic pregnancies. A single progesterone value could differentiate between ectopic and viable pregnancy in more than 80% of patients. The combination of all four biochemical markers predicted ectopic pregnancy with 98.5% specificity and 94.5% accuracy. Clinical diagnosis was less than 75% accurate. CONCLUSION: A combination of biochemical markers including hCG, progesterone, E2, and AFP can be superior to a single progesterone level or clinical evaluation in the diagnosis of ectopic pregnancy