Interleukin‐1 (IL‐1) has been implicated in the mechanism of human parturition in the setting of infection. The purpose of this study was to determine the effect of labor (term and preterm) and microbial invasion of the amniotic cavity on amniotic fluid (AF) concentrations IL‐1 α and IL‐1 β. AF was retrieved by transabdominal amniocentesis from the following groups of women: midtrimester genetic amniocentesis (16 to 18 wk) (N = 15), preterm labor with intact membranes (21 to 36 wk) with or without infection (N = 72), preterm premature rupture of membranes (PROM) (N = 88), and term not in labor or in active labor with or without infection (N = 58). AF was cultured for aerobic and anaerobic bacteria as well as Mycoplasmas. IL‐1 was measured with a commercially available immunoassay validated for AF (sensitivity: IL‐1α, 157 pg/ml; IL‐1β, 50 pg/ml). All women at midtrimester had undetectable AF IL‐1α and IL‐1 β. Among women in preterm labor with positive AF cultures, IL‐1α and IL‐1 β were detectable in the AF in 86.6% (13/15) and 100% (15/15), respectively. In contrast, all women with negative AF cultures without labor (N = 36) had undetectable AF IL‐1α concentrations and 52.7% (19/36) had undetectable AF IL‐1β concentrations. Histopathological chorioamnionitis was present in 92.8% (13/14) of patients who had positive AF cultures and detectable IL‐1 in the AF. IL‐1 was significantly higher in patients with preterm PROM, labor, and positive AF cultures than in the other subgroups of patients with preterm PROM. Among women at term who were not in labor, 26.3% (5/19) had detectable IL‐1α and 19.8% (3/19) had detectable IL‐1β in the AF. Among those in active labor with positive AF cultures, 44.4% (4/9) had detectable IL‐1α and 88.8% (8/9) had detectable IL‐1β in the AF. IL‐1β was detected more frequently in patients at term with positive AF cultures than in those with negative AF cultures (88.8% [8/9] vs. 38.7% [19/49]; P = 0.0088). We conclude that AF IL‐1 concentrations are elevated in women with preterm labor an microbial invasion of the amniotic cavity and in women with spontaneous parturition at term.