The tumor antigen 90K (Mac-2BP, L3 antigen), which has been shown to have T cell costimulatory activity, is a approximately 90 kDa secreted protein found in high levels in plasma, saliva, breast milk and other human fluids. The 90K antigen can be divided into three domains: an amino terminal scavenger receptor cysteine-rich (SRCR)-like domain (D1), followed by a heavily glycosylated mucin-like domain (D2) and a approximately 27 kDa carboxy-terminal domain (D3). In this study we report on the construction of six different 90K immunoglobulin (Ig) fusion proteins containing different 90K domain combinations. Initially these fusion proteins were used to identify which 90K domain contains the epitopes recognized by the anti-90K monoclonal antibodies (mAb) SP2 and L3. Both of these mAbs were found to recognize 90K-D2. A new panel of anti-90K mAb was then generated by immunizing mice with ascites derived 90K protein. The 90K domain specific fusion proteins were then used to identify novel anti-90K mAbs which recognize the amino terminal SRCR domain and the carboxy terminal approximately 27 kDa domain of 90K. Two novel anti-90K SRCR (D1) and one anti-90 27 kDa domain (D3) mAbs were obtained. These 90K-Ig fusion proteins, as well as the novel and existing anti-90K mAbs, provide a set of tools which will allow further dissection of the structure and function of this immune modulatory protein.