Our previous studies have shown that isolated cytotoxic T lymphocyte (CTL), B-cell, and T-helper epitopes, for which we coined the term minigenes, can be effective vaccines; when expressed from recombinant vaccinia viruses, these short immunogenic sequences confer protection against a variety of viruses and bacteria. In addition, we have previously demonstrated the utility of DNA immunization using plasmids encoding full-length viral proteins. Here we combine the two approaches and evaluate the effectiveness of minigenes in DNA immunization. We find that DNA immunization with isolated minigenes primes virus-specific memory CTL responses which, 4 days following virus challenge, appear similar in magnitude to those induced by vaccines known to be protective. Surprisingly, this vigorous CTL response fails to confer protection against a normally lethal virus challenge, although the CTL appear fully functional because, along with their high lytic activity, they are similar in affinity and cytokine secretion to CTL induced by virus infection. However this DNA immunization with isolated minigenes results in a low CTL precursor frequency; only 1 in approximately 40,000 T cells is epitope specific. In contrast, a plasmid encoding the same minigene sequences covalently attached to the cellular protein ubiquitin induces protective immunity and a sixfold-higher frequency of CTL precursors. Thus, we show that the most commonly employed criterion to evaluate CTL responses-the presence of lytic activity following secondary stimulation-does not invariably correlate with protection; instead, the better correlate of protection is the CTL precursor frequency. Recent observations indicate that certain effector functions are active in memory CTL and do not require prolonged stimulation. We suggest that these early effector functions of CTL, immediately following infection, are critical in controlling virus dissemination and in determining the outcome of the infection. Finally, we show that improved performance of the ubiquitinated minigenes most probably requires polyubiquitination of the fusion protein, suggesting that the enhancement results from more effective delivery of the minigene to the proteasome.