We assessed the costs and effectiveness of rapid HIV testing services provided to transgender communities in New York City and San Francisco from April 2005 to December 2006. Program costs were estimated based on service provider's perspective and included the costs attributable to staff time, incentives, transportation, test kits, office space, equipment, supplies, and utilities. The average annual numbers of persons tested were 195 and 106 persons and numbers notified of new HIV diagnoses were 35 (18.2%) in New York City and 8 (7.3%) in San Francisco, respectively. The estimated annual program costs were $125,879 and $64,323 and average costs per person notified of new diagnosis were $3,563 and $8,284 in New York City and San Francisco, respectively. The primary reason for differences in program costs by site was differences in the proportion of undiagnosed HIV infection among persons tested. Our findings can inform decisions about program planning and allocation of limited HIV testing resources.