We evaluated the accretion of 131I-labeled NP-4 anticarcinoembryonic antigen (CEA) into 4 size-matched human colonic carcinomas grown s.c. in nude mice. Antibody uptake for LS174T and GW-39 tumors was relatively high (19 to 23% ID/g on day 3), whereas moderate uptake was seen in the Moser tumor (7.5% on day 3) and low uptake was detected in the GS-2 tumor (1.8% on day 3). Blood clearance of radioantibody was twice as fast in mice with GS-2 tumors than in mice with GW-39, LS174T or Moser tumors. Seven physiological parameters that might influence radioantibody accretion were evaluated in order to better understand the differences in observed tumor targeting: vascular volume, blood flow rate, vascular permeability, tumor antigen content, serum antigen content and complexation of radioantibody, intratumoral antigen distribution, and intracellular antigen distribution. Although marked variability in vascular physiology, antigen content and antibody complexation of the 4 tumors grown in the same host and site existed, it was insufficient to explain the differences in antibody uptake. However, intra-tumoral distribution of antigen, and sub-cellular accessibility of antigen for radioantibody were important considerations. GS-2 tumors are well differentiated and have polarized cells. CEA in GS-2 is largely inaccessible to radioantibody; most of the antigen is located in the lumen of the glands or on the apical surface of gland cells and most of the antibody distributes to the stromal region on the basolateral surface. The low antibody targeting in GS-2 could therefore be explained by restricted intra-tumor accessibility of antibody. Scatchard analysis of NP-4 binding to Moser cells under non-internalizing and internalizing conditions revealed that 90% of the antigen is found within the cell, unavailable to bind with the NP-4 antibody, which is slow to internalize. In contrast, CEA in LS174T cells was almost entirely accessible. The reduced antibody targeting to Moser xenografts might therefore, be explained by restricted antibody accessibility at the cellular level.