Hidden anti-nuclear antibodies are demonstrated by immunofluorescence using smears of rat nuclei as substrate and rat liver section technique when sera are incubated with penicillamine. The non-detection of hidden anti-nuclear antibodies by tissue sections in the absence of a splitting agent may be due to the formation of high molecular weight complexes between rheumatoid factors and anti-nuclear antibodies. These high molecular weight complexes containing anti-nuclear antibodies do not have access to tissue nuclear antigens, but can react directly with free nuclei. It is postulated that anti-nuclear antibodies may represent the early pathway of both rheumatoid arthritis and connective tissue diseases. The demonstration of hidden anti-nuclear antibodies in seropositive sera indicates that rheumatoid factors may have a protective effect. It may explain dissimilarities observed in the clinico-immunological profile of rheumatoid arthritis and systemic lupus erythematosus. The splitting effect of penicillamine observed in vitro may be similar in vivo. It can explain clinical improvement and immunological side effects observed in rheumatoid arthritis patients treated with this drug.