Electron microscopic examination of SV40 chromatin prepared 44 hr post-infection led to the visualization of a nucleosome-free region (gap) in 15-20% of the minichromosomes. Minichromosomes with and without a gap displayed a mean number of 24 nucleosomes. Measurements carried out on dark field micrographs yielded for the gap a mean length of 249 +/- 13 bp, with a maximum value of 385 bp. The gap was mapped following digestion with three single-cut restriction endonucleases: Bgl l, Bam HI and Eco RI. It was located in the region of the origin of replication in accordance with previous biochemical data. To assess the situ existence of a nucleosome-free region, nuclei from infected cells were digested with DNase I. A highly sensitive region was thus revealed and mapped by secondary digestion with Eco RI. It was located in the same region as the gap, between 0.67 and 0.74 on the physical map. The sensitive region could be detected throughout the late phase of the virus cycle. These findings strongly suggest that a nucleosome-free region exists in the cells. The gap is not likely to be involved in replication, since it is asymmetric with respect to the Bgl I cleavage site, from which replication proceeds symmetrically.