The purpose of this study was to examine the relationship between mood and hormonal responses to cholinergic challenge with physostigmine in order to assess cholinergic system responsiveness in borderline personality disorder (BPD) patients, other non-BPD personality disorder patients, and normal controls. Thirty-four personality disorder patients, 10 of whom met criteria for BPD and 24 of whom met criteria for other, non-borderline, personality disorders, and 11 normal controls participated in a double blind, placebo controlled physostigmine challenge paradigm. The Profile of Mood States depression subscale (POMS-D) self report measure was obtained at baseline and following the physostigmine or placebo infusions. A repeated measures ANOVA of POMS-D scores in placebo and drug conditions indicated a significantly greater depressive response in the total cohort of personality disorder patients than in the normal comparison group (p < 0.05). However, the depressive response to physostigmine was significantly greater in BPD patients, but not other personality disorder patients, compared to normal controls (p < 0.05). There was a correlation between the peak placebo-corrected depressive response to physostigmine and a group of BPD traits related to affective instability but not a group of BPD traits related to impulsivity. There was no correlation in any group between mood response to physostigmine and changes in plasma cortisol, prolactin, or growth hormone, or to nausea or other side effects following physostigmine infusion. These data suggest that there is an association between BPD and acute depressive responses to physostigmine challenge, and that the cholinergic system may be involved in the regulation of affect in Axis II disorders.