Aims This study analyzed service unit and annual costs of substance abuse screening, brief intervention, and referral to treatment (SBIRT) programs implemented in emergency department (ED), inpatient, and outpatient medical settings in three U.S. states and one tribal organization. Methods Unit costs and annual costs were estimated from the perspective of service providers. Data for unit costs came from 26 performance sites, and data for annual costs came from 10 programs. A bottom-up approach was used to derive unit costs and included labor, space, and materials used in each SBIRT activity. Activities included direct SBIRT services and activities that support direct service delivery. Labor time spent in each activity was collected by trained observers using a time-and-motion approach. A top-down approach used cost questionnaires completed by program administrators to calculate annual costs and included labor, space, contracted services, overhead, training, travel, equipment, and supplies and materials. Costs were estimated in 2012 U.S. dollars. Results Average unit costs for prescreening, screening, brief intervention, brief treatment, and referral to treatment were $0.61, $6.59, $10.48, $22.63, and $12.06 in ED; $0.86, $6.33, $9.07, $27.61, and $8.03 in inpatient; and $0.84, $3.98, $7.81, $27.94, and $9.23 in outpatient settings, respectively; over half of the costs were attributable to support activities. Across all settings, the average cost to provide SBIRT per positive screen, for 1year, was about $400. Conclusions Support activities comprise a large proportion of costs. Health administrators can use the results to budget and compare how much sites are reimbursed for SBIRT to how much services actually cost.