Cytochalasin B (CB) was prepared by methanol extraction of dehydrated mold (Drechslera dematioidea) matte, reverse-phase C18 silica gel batch adsorption, selective elution with 1:1 (v/v) hexane:tetrahydrofuran (THF), crystallization, preparative TLC, and recrystallization. Unit gravity silica gel normal phase chromatography afforded additional CB. Yield per liter of medium was 300 mg of CB greater than 95% pure by NMR, HPLC (60:40 hexane:THF, Lichrosorb Si60 silica gel, 230 nm), and TLC. CB added exogenously to mouse organs at 1 and 5 micrograms/organ was recovered 70 to 100% by methanol extraction, adsorption to C18 silica gel Sep-Pak cartridges, elution with ethyl acetate, and analysis by TLC and/or HPLC. Limiting sensitivity (micrograms/extract) was 0.5 TLC; 1.0 HPLC. Quantitative extraction was confirmed with 3H-labeled CB. CB ip in mice at 50 mg/kg (LD10) distributed rapidly into liver, renal fat, kidney, intestines, mesentery, pancreas, spleen, and blood cells and was cleared from all but liver within 24 h. CB was below detectable levels in thymus, lymph nodes, heart, brain, bone marrow, and lungs. Cytochalasin A is fixed to tissues and not extractable. This work affords a source of CB in quantities permitting in vivo study, provides methods for extraction and analysis, and reveals the pharmacokinetics of ip bolus CB.