NY‐ESO‐1, a member of the CT (cancer/testis) family of antigens, is expressed in normal testis and in a range of human tumor types. Knowledge of NY‐ESO‐1 expression has depended on RT‐PCR detection of mRNA and there is a need for detecting NY‐ESO‐1 at the protein level. In the present study, a method for the immunochemical detection of NY‐ESO‐1 in paraffin‐embedded tissues has been developed and used to define the expression pattern of NY‐ESO‐1 in normal tissues and in a panel of human tumors. No normal tissue other than testis showed NY‐ESO‐1 reactivity, and expression in testis was restricted to germ cells particularly spermatogonia. In human tumors, the frequency of NY‐ESO‐1 antigen expression corresponds with past analysis of NY‐ESO‐1 mRNA expression e.g., 20–30% of lung cancers, bladder cancers and melanoma, and no expression in colon and renal cancer. Co‐typing of NY‐ESO‐1 antigen and mRNA expression in a large panel of lung cancers showed a good correlation. There is great variability in NY‐ESO‐1 expression in individual tumors, ranging from an infrequent homogeneous pattern of staining to highly heterogeneous antigen expression. © 2001 Wiley‐Liss, Inc.