OBJECTIVES/GOALS: Buprenorphine (BUP) is used for opioid use disorder during pregnancy but causes neonatal opioid withdrawal syndrome (NOWS). The goal of this study was to determine the contribution of the active metabolite, norbuprenorphine (NorBUP), to the development of NOWS when the parent drug, BUP, is administered during pregnancy. METHODS/STUDY POPULATION: Subcutaneously implanted osmotic minipumps delivered BUP (0, 0.01, 0.1 or 1 mg/kg/day) ± NorBUP (1 mg/kg/day) to pregnant Long-Evans rats from gestation day 9 until after delivery. NOWS was measured between 3 and 12 hours after delivery. Withdrawal was precipitated by an intraperitoneal injection of a mu opioid receptor antagonist naltrexone (NTX; 0, 1 or 10 mg/kg), and movement duration (MD; a validated proxy for NOWS) was measured using Noldus Ethovision. Concentrations of BUP, NorBUP, and their glucuronide conjugates in the brains of neonatal littermates not undergoing withdrawal testing were determined using LC/MS/MS. Two-way ANOVA and multiple linear regression analyses tested for interactions between BUP and NorBUP on MD and related brain concentrations to MD, respectively. RESULTS/ANTICIPATED RESULTS: There was no interaction effect between BUP and NorBUP on MD for either sex or at any dose of naltrexone. In females, but not males, BUP (1 mg/kg/day) significantly increased NorBUP-induced MD by 58% following an injection with 1 mg/kg NTX. A multiple linear regression model that included BUP and NorBUP brain concentrations as predictors of MD was significant and well-fitting [FEMALES: F (2, 40) = 23.97, P < .0001, adj R2 = 0.52; MALES: F (2, 40) = 5.84, P = .0059, adj R2 = 0.19]. There was a differential contribution of NorBUP brain concentrations to MD based on sex. The partial regression coefficient for NorBUP was 51.34 (p < .0001) for females and 19.21 (p = 0.093) for males. The partial regression coefficient for BUP was similar for females and males (FEMALES:βBUP = 10.62, p = .0017; MALES:βBUP = 11.38, p = .009). DISCUSSION/SIGNIFICANCE: We show for the first time a differential contribution of NorBUP to BUP-associated NOWS in each sex, suggesting sex differences in NorBUP susceptibility and implicating that treatment strategies reducing prenatal NorBUP exposure may be more effective for females than males.