Transforming growth factor-beta (TGF-beta) is an important regulator of development. In vitro, TGF-beta is secreted in a latent, inactive form and can be activated by pH extremes, chaotropic agents, or cell-surface proteases. However, there is little evidence for the existence of latent TGF-beta in vivo. In this study, we determined whether (1) cultured embryonic cardiac segments secrete latent or active TGF-beta, (2) binding of TGF-beta antibody to TGF-beta was conformation-dependent (i.e., active vs. latent), and (3) immunostaining of embryonic hearts changed after exposure to activating conditions. Only latent TGF-beta 3 (acid activatable) was detected in conditioned medium of stage 14-16 chick cardiac segments as measured by a growth inhibition bioassay. No growth-inhibitory activity was present in nonacidified control medium. When blotted onto a membrane, only transiently acidified conditioned medium bound TGF-beta antibody. These data showed that cardiac segments secrete latent TGF-beta which binds with antibody if activated. To determine if antibody binding to tissue sections required exposure to TGF-beta-activating conditions, stage 14-16 embryos were fixed and sectioned under conditions that maximally retained extracellular matrix (ECM). Under these conditions, immunostaining was found in the myocardium but not in the endocardium or cardiac ECM. Limited immunostaining was found in other areas of the embryo and was always cell-associated. In addition to the above staining, when tissue sections were exposed to TGF-beta activating conditions, immunopositive staining was present within most of the embryonic ECM including the cardiac ECM. All immunostaining was blocked by preabsorption with TGF-beta 3 protein. These data suggest that active TGF-beta has a very limited distribution while latent TGF-beta is more abundant in embryonic ECM. Therefore, in vivo activation of TGF-beta may play an important role in mediating the expression of TGF-beta function during development.