Rationale and objectives: Musculoskeletal (MS) symptoms are reported to affect up to 50% of women on AI and may lead to poor medication adherence. Vitamin D3 supplementation may decrease the MS pain, stiffness and weakness reported by women treated with AIs. We are conducting a controlled clinical trial (D3AI study) to assess the efficacy of vitamin D3 supplements in decreasing these symptoms and to examine the effects of vitamin D3 on the pharmacokinetics of AIs and on AI medication adherence. Methods: To test the effects of a daily dose of 4000IU D3 when compared to the usual care of 600IU D3 over a 7 month study period, 371 post-menopausal women on AIs were screened for MS symptoms using the average of = 1.5 on the musculoskeletal subscale questions of the Breast Cancer Prevention Trial symptoms scale (BCPT-MS). Subjects meeting inclusion criteria who consented to study participation (n = 48) received 600IU D3 during a 1 month run-in period prior to randomization. Following this run-in period, baseline serum 25(OH)D was assayed by a chemiluminescent immunoassay (Liaison analyzer, DiaSorin). AI adherence diaries were completed during the 1 month run-in and population pharmacokinetics (PK) studies were performed on the first participants enrolled for anastrozole (n = 8) and letrozole (n = 10) at baseline (after 1 month on 600IU D3) and again at 6 months after randomization to either 600IU or 4000IU D3. For PK studies, plasma samples were collected pre- AI dose and at 2- and 4 hr post AI dose and analyzed with validated tandem mass spectrometry (LC-MS/MS). PK parameters for each drug were determined by non-linear mixed effects modeling (NONMEM) and one-compartment models with first-order absorption were used to describe the plasma concentration-time data. Results: Thirty percent (n = 112/371) of subjects screened met the inclusion criteria of experiencing MS symptoms (BCPT-MS =1.5) and data on the first 48 subjects randomized are presented. BCPT-MS score at screening was 2.48 ± 0.62 (mean±sd). At baseline (following one month run-in period of 600 IU D3) BCPT-MS was 2.41±0.65 (mean±sd). Baseline serum 25(OH)D was 36±11 ng/ml (mean±sd) and AI adherence was 99% during this 30 day run-in period. Inter-individual variability of AI pharmacokinetic properties ((% CV) estimates of apparent AI clearance (CI/F) for anastrozole was 25% and for letrozole, 43.1%. Conclusions: AI-associated MS symptoms did not affect AI adherence in this population during the 30 day run-in period. Daily vitamin D3 supplements of 600IU for 30 days was adequate to maintain serum 25(OH)D levels of >30 ng/ml. The approximate 3 to 4-fold inter-individual variability in AI drug clearance may have a clinically meaningful impact on prevalence of symptoms, serum estrogen concentrations and treatment effica cy.