It has been suggested that residual cytotoxic sodium dodecyl sulfate (SDS) is responsible for the low levels of cell in-growth observed in SDS decellularized tissues. To determine whether this is the case, we used 2 washing methods to remove residual SDS and extensive biochemical, mechanical, and structural analyses to determine the effects of SDS-based decellularization on porcine anterior cruciate ligament (ACL) tissue and its propensity for cellular repopulation. The level of residual SDS in decellularized tissue was reduced using 2 different washing techniques (pH = 9 buffer, 75% ethanol). After washing in pH = 9 or 75% ethanol, residual SDS concentrations in decellularized tissues were found to be approximately 8 and 23 times less than reported SDS cytotoxic levels, respectively. It was found that SDS treatment significantly reduced glycosaminoglycan levels, increased collagen crimp amplitude and periodicity, and increased susceptibility of collagen to degradation by the gelatinase enzyme trypsin. The level of repopulation and viability of autologous ACL fibroblasts in the decellularized tissue after 28 days of culture were found to be the same regardless of the washing technique and resulting level of residual SDS in the tissue. This strongly indicates that alterations in tissue matrix biochemistry or structure from SDS treatment and not residual SDS cytotoxicity are responsible for the low cell re-population observed in SDS decellularized tissues.