ACTH, like other anterior pituitary peptide hormones, is secreted episodically and demonstrates both circadian and ultradian rhythms. CRH is the major regulator of ACTH release from the pituitary corticotroph. To determine the dependence of ACTH ultradian rhythms on CRH, passive immunoneutralization was used to block the activity of endogenous CRH in rats with indwelling venous catheters. Blood was sampled at 2- and 15-min intervals while blood volume was replaced. Plasma ACTH was measured by RIA. Time-series analysis of plasma ACTH concentrations was performed with PULSAR and Cluster Analysis. The 2 min data demonstrated secretory bursts approximately every 20 min. CRH immunoneutralization had no effect on the frequency of these pulses, but significantly reduced their amplitude. This was the case for raw data as well as data in which lower frequency variation had been filtered out. The 15 min data demonstrated pulsatile secretion, with a secretory episode approximately every 100 min. This lower frequency rhythm was also observed when high frequency components were filtered out of the 2 min data series. Analysis of the 15 min and the filtered 2 min time series showed this rhythm to be almost totally ablated by CRH immunoneutralization. These results suggest that CRH is responsible for amplitude modulation of an underlying CRH-independent rhythm and that through intermittent amplitude modulation of this rhythm a lower frequency rhythm is generated. Comparison between treatment groups of pulses identified by PULSAR or Cluster Analysis yielded similar results, but the programs were discordant with each other. This is the first in vivo evidence of pulsatile ACTH secretion independent of CRH, the first report demonstrating that different ultradian rhythms of ACTH may be regulated by different mechanisms, and the first comparison of PULSAR and Cluster Analysis on plasma ACTH time series.