We have previously reported the sequential appearance in hyperimmunized rabbits of three distinct types of antifluorescein-combining sites that can be distinguished by the characteristic induced circular dichroism (CD) of the bound hapten, fluorescein. Such induced CD depends on the configuration of the surrounding residues, and, in the case of anti-fluorescein, can be localized to the configuration of the sub-site which binds the hydroxyxanthenone moiety of fluorescein. In the present investigation, we have studied the fine structure of both autologous and heterologous recombinant sites and of the free chains by measuring the induced CD of bound hapten. Heavy chain dimers at pH 5.4 bound fluorescein that displayed a weak negative CD band which is different from that observed in any of the native antibodies. No induced CD was observed with light chains. Thus, the hydroxyxanthenone subsite does not exist intact in any of the isolated chains. Fluorescein bound to purified autologous recombinants, when studied at pH 7.5, exhibited CD spectra very similar to those observed for the original antibodies. Most significantly, fluorescein bound to purified heterologous recombinants, prepared in all possible combinations, showed CD spectra most similar to those exhibited by sites from which the heavy chain was derived. Thus, the microenvironment of the subsite appears to be due primarily to the heavy chain.