Intracytoplasmic sperm injection (ICSI) of DNA-loaded sperm cells has been shown to be a valuable tool for the production of transgenic animals, especially when DNA constructs with submegabase magnitude are used. In order to optimize and to understand the mechanism of the ICSI-mediated transgenesis, we have evaluated the impact of transgene DNA concentration, transgene flanking with nuclear matrix attachment regions (MARs), and the use of recombinase A (RecA)-coated DNA on the efficiency of mouse transgenesis production by ICSI. Presented data include assays with three DNA constructs; an enhanced green fluorescent protein (EGFP) plasmid of 5.4 kb, this plasmid flanked with two MAR elements (2.3 Kb of the human beta-interferon domain boundaries), and a yeast artificial chromosome (YAC) construct of ~510 kb (the largest transgenic construct introduced by ICSI that we have seen reported). ICSI-mediated transgenesis was done in the B6D2 mouse strain using different concentrations for each construct. Analysis of generated data indicated that ICSI allows the use of higher DNA concentrations than the ones used for pronuclear microinjection, however, when a certain threshold is exceeded, embryo/fetal viability decrease dramatically. In addition, independently of the transgene concentration tested, transgene flanking with MAR sequences did not have a significant impact on the efficiency of this transgenesis method. Finally, we observed that although the overall efficiency of ICSI-mediated transgenesis with fresh spermatozoa and RecA-complexed DNA was similar to the one obtained with the common ICSI-mediated transgenesis approach with frozen-thawed spermatozoa and RecA free DNA, this method was not as efficient in maintaining a low frequency of founder animal mosaicism, suggesting that different mechanisms of transgene integration might result from each procedure.