The accurate estimation of gestational age in field studies in rural areas of developing countries continues to present difficulties for researchers. Our objective was to determine the best method for gestational age estimation in rural Guatemala. Women of childbearing age from four communities in rural Guatemala were invited to participate in a longitudinal study. Gestational age at birth was determined by an early second trimester measure of biparietal diameter, last menstrual period (LMP), the Capurro neonatal examination and symphysis-fundus height (SFH) for 171 women-infant pairs. Regression modelling was used to determine which method provided the best estimate of gestational age using ultrasound as the reference. Gestational age estimated by LMP was within +/-14 days of the ultrasound estimate for 94% of the sample. LMP-estimated gestational age explained 46% of the variance in gestational age estimated by ultrasound whereas the neonatal examination explained only 20%. The results of this study suggest that, when trained field personnel assist women to recall their date of LMP, this date provides the best estimate of gestational age. SFH measured during the second trimester may provide a reasonable alternative when LMP is unavailable.