The aim of the present experiment was to investigate the effect of [D-Met2,Pro5] enkephalinamide (ENK) implantation on the development of the uterus during 8-33 days of age and the involvement of epidermal growth factor (EGF) in the effect. Administration of ENK was attained by osmotic minipumps (5 microg/h) implanted intraperitoneally. ENK resulted in a decrease in the EGF content of the uterus, which was already significant after 48 h of the implantation. The DNA content 24 and 48 h after the treatment decreased, no change at 72 h was found, however the protein/DNA ratio on the effect of ENK treatment was significantly decreased at this time in all examined age groups. High affinity and lower capacity competitive naloxone binding sites were demonstrated in the membrane fraction of the uteri. Seventy-two h after ENK treatment the binding capacity of these sites significantly dropped. The present results suggest a novel multiple interaction between estrogen and two probably paracrine hormones, EGF and opioid peptide, in the regulation of growth and development of the uterus.