We have previously described a 3.5-Mb domain of enhance scaffold/matrix attachment region (S/MAR) at a human neocentromere, and normal expression of underlying genes within this region. We also reported that partial inhibition of histone deacetylation using 33 nmtrichostatin A (TSA) resulted in a shift in the position of the CENP-A-binding domain within the neocentromere, with no noticeable effects on mitotic segregation function. In this study, 33 nM TSA caused a reduction in the size of the enhanced S/MAR domain of one-half to 1.7 Mb. Treatment with a DNA-intercalating drug distamycin A (DST) at 75 microg/ml resulted in a size reduction of the enhanced S/MAR domain at the neocentromere of two-thirds to 1.2 Mb, and that of the CENP-A-binding domain of 40%, from 330 to 196 kb, with no significant shift in the position of the latter domain. Other DST effects include mitotic chromosomal missegregation, reduction in the levels of Topo IIalpha, CENP-A, CENP-C, and HP1alpha, and an increase in mitotic checkpoint protein BubR1. TSA or DST treatment similarly resulted in a significant reduction, by approximately 20 and 50%, respectively, in the size of the enhanced S/MAR domain at the alpha-satellite DNA of a native chromosome 10 centromere. Transcriptional competence within the neocentromere is overall not noticeably altered by either TSA or DST treatment, as is evident from the absence of any significant increase or decrease in the expression levels of 47 underlying genes tested. These results suggest that a substantial contraction of the S/MAR domain may not be deleterious to centromere function, that disruption of the S/MAR domain directly affects the binding properties of a host of scaffold/matrix and centromeric/pericentric proteins, and that the overall competence and regulation of transcription at the neocentromeric chromatin is similar to those found at the corresponding normal genomic sites.