A plasmid DNA containing a chimeric immunoglobulin heavy-chain gene with tissue-specific promoter and enhancer elements was used as a model system to study the events triggered by a single intraspleen DNA inoculation in adult C57Bl/6 mice. A single intraspleen inoculation was followed in a week by secretion of transgene immunoglobulins and production of immunoglobulin M (IgM) anti-immunoglobulins. Their kinetics of serum appearance were almost superimposable. While anti-immunoglobulin antibodies remained detectable for over 6 months, transgene immunoglobulins disappeared after 3-4 weeks. However, transgene mRNA was detected in the spleen for 4 months. A multiplex polymerase chain reaction (PCR) analysis on splenic genomic DNA 17 days after inoculation demonstrated that the transgene was integrated in the host chromosomal DNA. The nucleotide sequence of the rearranged VDJ region from splenic genomic DNA was identical to that of the parental plasmid DNA, hence ruling out that hypermutation had occurred. A booster injection of immunoglobulin encoded by the transgene on day 200 elicited a typical secondary immune response with IgG1 and IgG2b antibodies. These results demonstrate that a single inoculation of an immunoglobulin heavy-chain DNA targeted to spleen lymphocytes leads to spontaneous integration of the transgene into the host DNA, and that this is sufficient to initiate immunity and establish immunologic memory. Our data also show that minute amounts (<100 ng/ml) of an endogenously produced protein secreted in the microenvironment of a lymphoid tissue generate immunity and establish immunologic memory rather than tolerance.