We considered that accumulation of nonesterified (free) fatty acids (NEFAs) in the first trimester of pregnancy would mark women at excess risk of spontaneous preterm birth (sPTB) and examined the interplay between NEFAs, lipids, and other markers to explore pathways to sPTB. In a case-control study nested in the Pregnancy Exposures and Preeclampsia Prevention Study (Pittsburgh, Pennsylvania, 1997-2001), we assayed NEFA levels in nonfasting serum collected at a mean gestational week of 9.4 (range, 4-20 weeks) in 115 women with sPTB (<37 weeks) and 222 women with births occurring at ≥37 weeks. C-reactive protein, total cholesterol, low-density lipoprotein and high-density lipoprotein (HDL) cholesterol, triglycerides, and uric acid were also measured. Polytomous logistic regression models were used to evaluate tertiles of NEFA levels and sPTB at <34 weeks and 34-36 weeks; factor analysis was used to characterize patterns of biomarkers. Women with NEFA levels in the highest tertile versus the lowest were 2.02 (95% confidence interval: 1.13, 3.48) times more likely to have sPTB, after adjustment for covariates. Risk of sPTB before 34 weeks was particularly high among women with high NEFA levels (odds ratio = 3.73, 95% confidence interval: 1.33, 10.44). Six biomarker patterns were identified, and 2 were associated with sPTB: 1) increasing NEFA and HDL cholesterol levels and 2) family history of gestational hypertension. NEFA levels early in pregnancy were independently associated with sPTB, particularly before 34 weeks. We also detected a novel risk pattern suggesting that NEFAs together with HDL cholesterol may be related to sPTB.