Integrative and conjugative elements (ICEs) are widespread mobile DNA within bacterial genomes, whose lifestyle is relatively poorly understood. ICEs transmit vertically through donor cell chromosome replication, but in order to transfer, they have to excise from the chromosome. The excision step makes ICEs prone to loss, in case the donor cell divides and the ICE is not replicated. By adapting the system of LacI-cyan fluorescent protein (CFP) binding to lacO operator arrays, we analyze here the process of excision and transfer of the ICE for 3-chlorobenzoate degradation (ICEclc) in individual cells of the bacterium Pseudomonas putida. We provide evidence that ICEclc excises exclusively in a subset of specialized transfer-competent cells. ICEclc copy numbers in transfer-competent cells were higher than in regular nontransferring cells but were reduced in mutants lacking the ICE oriT1 origin of transfer, the ICE DNA relaxase, or the excision recombination sites. Consistently, transfer-competent cells showed a higher proportion without any observable LacI-CFP foci, suggesting ICEclc loss, but this proportion was independent of the ICE relaxase or the ICE origins of transfer. Our results thus indicated that the excised ICE becomes transiently replicated in transfer-competent cells, with up to six observable copies from LacI-CFP fluorescent focus measurements. Most of the observed ICEclc transfer to ICE-free P. putida recipients occurred from donors displaying 3 to 4 ICE copies, which constitute a minority among all transfer-competent cells. This finding suggests, therefore, that replication of the excised ICEclc in donors is beneficial for transfer fitness to recipient cells. IMPORTANCE Bacterial evolution is driven to a large extent by horizontal gene transfer (HGT)-the processes that distribute genetic material between species rather than by vertical descent. The different elements and processes mediating HGT have been characterized in great molecular detail. In contrast, very little is known on adaptive features selecting HGT evolvability and fitness optimization. By studying the molecular behavior of an integrated mobile DNA of the class of integrative and conjugative elements in individual Pseudomonas putida donor bacteria, we report here how transient replication of the element after its excision from the chromosome is favorable for its transfer success. Since successful transfer into a new recipient is a measure of the element's fitness, transient replication may have been selected as an adaptive benefit for more-optimal transfer.