This study provides the first measurement of microplastic abundance and distribution in surface waters and sediments in Tampa Bay, FL. Microplastic concentrations in discrete water samples ranged from 0.25 to 7.0 particles/L with an average of 0.94 (±0.52) particles/L. Samples taken with a 330 μm plankton net had 1.2–18.1 particles/m3 with an average of 4.5 (±2.3) particles/m3. Discrete samples were 200 times higher than net samples, suggesting substantial losses or undersampling with the net. For both discrete and plankton tow samples, there were no significant differences in concentrations between stations or regions. Intense rainfall events in the summer always preceded samples with substantially higher counts. Most (>75%) microplastics were fibers. Using an average value of 1 particle/L, Tampa Bay contains ~4 billion microplastic particles. Surface sediments had an average of 280 (±290) particles/kg, ranging from 30 to 790 particles/kg. Highest concentrations of microplastics were found in sediments close to industrial sources; lowest values in Middle and Lower Tampa Bay are consistent with shorter residence times.