Amphotericin B formulations were compared in preclinical models by using intraperitoneal (ip) and intravenous (iv) delivery of amphotericin B deoxycholate (DAMB) or liposomal amphotericin B. We examined the effects on drug tissue penetration and retention resulting from different routes of drug administration. Mice were treated with equivalent total doses of AmBisome (AmBi) or DAMB (i.e.,15 mg/kg) given ip (3 mg/kg/day for 5 days) or iv (3 mg/kg/day AmBi for 5 days or 1 mg/kg/day DAMB for 15 days), with tissues collected 24 h post-treatment. For drug retention studies, mice were given iv or ip total doses of 30 mg/kg AmBi (10 mg/kg/day 3 x /week) or 60 mg/kg AmBi (20 mg/kg/day 3 x /week) with tissue collection 24 h or 7 days post-treatment. Blood samples were collected at 0.5 h, 2 h, 8 h, 12 h and 24 h after ip or iv drug dosing. A Paecilomyces variottii bioassay was used to determine drug concentrations. AmBi and DAMB were detected in the kidneys following iv, but not ip dosing. Significantly more DAMB than AmBi was detected in the lungs with ip dosing (P = 0.008), and more AmBi than DAMB (P = 0.056) was present with iv dosing. Unlike the lungs, the spleen and liver retained the AmBi for up to one week post-treatment regardless of the route of drug administration. Thus, there are significant differences in AmBi and DAMB tissue distribution depending upon the drug route and these differences could effect how the drugs perform in fungal infection models.