Initiation and maintenance of lactation are complex phenomena involving cellular and enzymatic changes including those in mammary gland and adipose tissue. Fat is the main energy component of the milk, originating from the diet, mobilization from adipocytes, and from de novo synthesis in the mammary gland. Milk fat synthesis is controlled in various ways and one example is via regulation of acetyl-CoA carboxylase α (ACCα), a key enzyme in the de novo fatty acid synthesis pathway. ACCα is encoded by mRNAs transcribed from 3 promoters (PI, PII and PIII) characterized as tissue-specific in the ovine genome. PI transcripts are restricted to the adipose tissue and liver, whereas PII is found in all tissues and PIII has been isolated from mammary tissue from lactating ewes. This study evaluated the transcription of ACCα from PI, PII and PIII in mammary and adipose tissue from dairy ewes at different stages of lactation. Mammary gland and adipose tissue biopsies were taken from 6 lactating ewes at 3 periods (15, 70 and 120 d in milk) to represent early, mid and late lactation. Subsequently RNA was extracted, cDNA synthesized and qRT-PCR analysis conducted. Data were analyzed by SAS using ribosomal protein S18 housekeeping gene as a covariate. As expected, no expression of PI was detected in the mammary gland. Transcripts from PII in mammary gland were higher in early lactation and decreased as lactation advanced (early vs. mid P = 0.02; early vs. late P = 0.002; mid vs. late P = 0.23). Similarly, the PIII transcripts decreased throughout lactation in mammary gland (early vs. mid P = 0.001; early vs. late P = 0.001; mid vs. late P = 0.005). On the contrary, in adipose tissue the PI gene expression was higher in mid and late lactation (P = 0.001), and transcripts from PII and PIII did not differ among periods. Results suggest that PII transcripts in mammary glands are upregulated in early and mid lactation according to a higher requirement for fat synthesis and PI transcripts have a greater expression level in adipose tissue in late lactation likely due to a positive energy balance replenishing body fat reserves.