The objective of the present study was to determine if the concentration of protoporphyrin 1X (PplX) in the rat endometrium could be increased by administering 5-aminolevulinic acid (ALA) in multiple doses or by continuous infusion. The effect of pH, temperature and time in solution on the stability of ALA were also investigated. Estrogen-filled silastic capsules were implanted subcutaneously into ovary intact female rats (200-225 g) (n =66). On the third day of hormonal priming, ALA ( 10 mg or 25 mg) dissolved in saline and adjusted ~o a pH of 5-5.5 was administered intrauterine either as a single bolus or as two injections 3 hours apart (n = 10). A fifth group of rats was infused with 25 mg ALA over a 12 hour period using an osmotic minipump ( n = 6). In a second experiment, ALA ( 25 mg ) was injected immediately after being dissolved in saline (pH 2) (n= 16) or after incubation at 37 Β°C for 12 hour (pH 2) (n=7). PplX was then extracted from the endometrium and myometrium using a I:1 methanol/perchloric acid solution and quantified spectrofluor(,metrically. A dose-response relationship was observed between 10 and 25 mg of ALA and endometrial PplX concentrations. However, no differences in endometrial PplX concentrations were detected between rats administered ALA either as a single bolus or as two doses. Continuous infusion of 25 mg of ALA resulted in statistically lower endometrial PplX concentrations compared to 25 mg ALA injected either as a single bolus or as two injections. Neither pH, temperature, nor time in solution affected ALA-induced PplX accumulation, We conclude that the simplest way of achieving the highest PplX concentration in the rat endometrium in vivo is to administer a bolus injection of 25 mg of ALA.