Cultivation of primary adult hepatocytes creates a challenge because of their loss of hepatocellular functions if prevented from attaining polarized cell-cell and cell-matrix interactions. Thus, when hepatocytes are seeded in nonadhesive porous alginate scaffolds, they form 100-μm-diameter spheroids with enhanced cell-cell interactions. Using transmission electron microcopy (TEM), histology, and functional studies, we investigated the state of hepatocyte spheroids during in vitro cultivation. TEM of day 3 spheroids revealed multiple cell layers, with tight junctions between adjacent cells and microvillus-lined channels that resembled bile canaliculi, both structurally and functionally. When copper ions were added to the external medium, the spheroidal hepatocytes performed endocytosis and eventually secreted the heavy metal ions into the bile lumens. From day 8 on, however, there was a rapid decline in cell viability. Histology and TEM analysis of day 13 spheroids revealed a necrotic center, with one viable cell layer on the outskirts. The absence of DNA laddering and negative results in TUNEL assay indicated that apoptosis is not the main process leading to cell death. Cell necrosis may be a result of accumulated bile secretions in the compacted spheroids. Collectively, our results suggest that spheroids derived from adult hepatocytes may have limited utility in long-term applications.