OBJECTIVE To evaluate the effects of time and storage temperature on amniotic fluid (AF) glucose concentration. METHODS Amniocentesis was performed on 47 patients with preterm labor or preterm rupture of membranes at or before 34 weeks' estimated gestation and the AF was cultured for aerobic, anaerobic, Mycoplasma, and Ureaplasma organisms. Twenty-one samples were stored at 37C, 13 were kept at room temperature (22C), and 13 were frozen (-20C). Glucose concentration was measured at 0 and 12 hours on all AF samples and at 2, 4, and 6 hours on the unfrozen samples. RESULTS No significant change in AF glucose concentration occurred over time at any storage temperature in the 35 samples with negative cultures. In 12 AF samples with positive cultures, a significant decrease from baseline was noted at 2 and 4 hours in those kept at 37C and 22C (P = .016). Glucose concentration was unchanged in frozen samples regardless of the culture results. The initial AF glucose concentration was significantly lower in samples with positive than in those with negative cultures (P < .0001). CONCLUSIONS Amniotic fluid glucose concentration does not change over 12 hours in the absence of intra-amniotic infection.