OBJECTIVES: To compare two different approaches to estimate the total cost Bolivia’s Expanded Program on Immunization (EPI), one using the central budget reports vs. an alternative approach using a novel ProVac EPI costing Tool. METHODS: The first approach was applied using cost data from the 2009 budget approved to the Bolivian Ministry of Heath and regional sources of wage assignments. We estimated the annual cost of Bolivian national EPI following the WHO recommendations for estimating cost of immunization programs. The second approach use a new EPI costing tool developed by the PAHO ProVac Initiative and designed to estimate the costs of every single component of a standard vaccination program at three administrative levels (district, state, national). A costing survey was carried out at different vaccination centers. RESULTS: The central level budgeting approach resulted in an overall EPI cost of US $11,960,000 for 2009. Using the EPI costing tool to assess the local and regional costs of vaccination produced an estimate four times higher, US $53,330,000. The most important items were the purchase of vaccines and vaccination supplies (46% of the total), followed by personnel who contributed about 28%. Cost incurred in the direct delivery of vaccine to patients were 50% of the total, with major participation of personnel and transportation items.  Other cost like vaccination supplies were best defined with this new approach and also explains the incremental difference. CONCLUSIONS: The central Budget cannot be relied on for a full estimation of EPI program costs, it can lose cost details within the same level.  Also, the costs incurred at lower administrative levels were not well-captured in the central budget and are important of total EPI costs. The costs of vaccination other than vaccine acquisition and vaccination supplies are very important and should be considered in cost analysis of vaccine introduction.