Objective: To assess mid-trimester amniotic fluid concentrations of interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-α, and lipopolysaccharide binding protein (LBP) in pregnancies with normal outcome and correlate them with gestational week (GW), parity, and fetal gender. Methods: Cytokine concentrations were measured within a week of amniocentesis during GW 15+0 to 20+6 and correlated with GW at birth, parity, and fetal gender. Results: After exclusion of women with an adverse pregnancy outcome or those lost to follow-up, 273 consecutive patients were evaluated (median parity: 1; range: 0–5). Ranges for IL-6, IL-8, TNF-α, and LBP were 4.9–2620 pg/mL, 36.2–5843 pg/mL, 8.0–28.2 pg/mL, and 0.06–1.9 μg/mL, respectively. IL-6, IL-8, and LBP values did not respectively differ among time points, but TNF-α values did between the 15 th and 16 th and the 15 th and 18 th weeks of gestation (P<0.05). No significant correlations between cytokine levels and parity or fetal gender were identified. Conclusions: Cytokine concentrations in amniotic fluid during the mid-trimester did not differ with parity or fetal gender. IL-6, IL-8, and LBP levels appeared stable with GW, whereas GW significantly influenced TNF-α concentrations. Further analyses are warranted to establish the role of cytokines in predicting adverse pregnancy outcomes.