The Mascot-Jefferson City district zinc deposits are of the Mississippi Valley-type (MVT), hosted by carbonate rocks and dominated by sphalerite mineralization in strata-bound breccia bodies. We have utilized the high spatial resolution (20-30 µm) of the ion microprobe to obtain in situ sulfur isotopic analyses from discrete growth zones in sphalerite and analyses of associated pyrite. Two types of pyrite were noted: pre-sphalerite, diagenetic pyrite (d³⁴S=-16.1‰ and -20.0‰) and syn-sphalerite pyrite that is intergrown with sphalerite (d³⁴S=31.3‰ to 33.7‰). Two textural varieties of sphalerite mineralization (zoned and unzoned) were characterized. Zoned sphalerite has d³⁴S values from 27.8‰ to 51.0‰, high Cd contents (up to 0.96 wt. %) and dark areas that are likely due to minute inclusions of organic carbon. The unzoned sphalerite has d³⁴S values from 20.2‰ to 39.5‰, high Fe content and no organic inclusions. Regardless of the textural variety of sphalerite mineralization, the sulfur isotopic composition of sphalerite is heterogeneous and can vary by as much as 15‰. The d³⁴S values recorded in this study are among the heaviest ever reported for MVT sphalerite. The micro-scale d³⁴S variations and presence of such high d³⁴S values have not been previously documented for east Tennessee. The data presented here suggests multiple sulfur sources and complex precipitation mechanisms, and several possible mechanisms are examined. The most probable scenario involves significant sulfur input from a sulfate- and metal-bearing fluid of variable d³⁴S composition mixing with a gas cap containing H₂S of relatively homogeneous d³⁴S composition. The gas cap provided lesser amounts of sulfur to the system. Mixing of two isotopically different sulfur sources of variable proportions can account for the observed microscale variation in d³⁴S (sphalerite).