The SITS (Safe Implementation of Thrombolysis in Stroke)-EAST project was launched in 2007 to support evidence-based stroke treatments, including thrombolytic treatments (TLTs), in Central and Eastern Europe. The aim of this study was to detail the topography, efficacy, and specific characteristics of stroke centers in the Czech Republic (CZ), the country with the highest rate of TLT. The nationwide study was performed between September and November 2007 through a questionnaire sent to all centers registered in SITS. The questionnaire contained 25 questions addressing how centers met recommendations for primary and comprehensive stroke centers in 2006. Center characteristics predicting the number of TLTs were identified by multiple regression analysis. Altogether, data were obtained from 42 centers (88% response rate) with the following characteristics [median (min-max)]: catchment area population 150,000 (90,000-750,000); number of strokes/year 420 (150-1,589); number of TLTs 7.5 (2-45); number of physicians specialized in stroke 3 (1-7). Centers treated 4.3% of the patients with ischemic strokes with tissue plasminogen activator. A stroke unit was present in 32 (78%) departments. Rescue interventional therapies were performed in 11 (26%). Financial problems restricted TLT in 14 (35%) centers. Fourteen factors were associated with the number of TLTs in univariate regression analysis, but in multiple regression analysis, only the catchment area population (p < 0.001), >3 physicians specialized in stroke (p < 0.001), and the presence of an acute interventional program (p < 0.006) remained significant. This study provides the fundamental stroke treatment network data that are essential for certification and further SITS-EAST TLT expansion.