It has been demonstrated that circulating kisspeptin is increased during the attainment of puberty in humans. To determine if hypothalamic (MBH) expression of kisspeptin (Kiss1), anterior pituitary (AP) expression of GnRH receptor (GnRHr) and LH-β, and MBH and AP kisspeptin content increase at pubertal onset in the gilt, the following experiment was performed. Twenty-four crossbred gilts of similar age (150 d) and weight (102.7 ± 0.3 kg) were relocated and exposed to a mature boar for estrus detection on d 1 and continuing for 6 d. Gilts that stood immobile within 24 h of slaughter (d 6) were considered to have attained puberty. Plasma samples were collected on d 1, 3, and 6. All gilts were slaughtered on d 6 when MBH, AP, and blood were collected. Relative expression of MBH Kiss1 and β-actin and AP GnRHr, LH-β, and β-actin was determined using real-time reverse transcriptase PCR. Fold changes in relative expression were determined using the Relative Expression software tool. Relative expression is based on the expression ratio of a target gene versus a reference gene. The expression ratio results of the transcripts were tested for significance by a pair-wise fixed reallocation randomized test with day compared as independent time effects. Hypothalamic and AP content of kisspeptin were determined by RIA and differences were determined using the MIXED procedure of SAS. Relative expression of Kiss1 was increased (P = 0.005) 2.2 fold in the gilts that had attained puberty. Relative expression of GnRHr did not differ (P > 0.05) in gilts that had attained puberty versus those that did not. Relative expression of LH-β tended to be decreased (P = 0.09) 0.80 fold in gilts that had attained puberty. AP concentrations of kisspeptin were not different (P > 0.05) between treatments. Kisspeptin content in the MBH was increased (P = 0.03) in gilts that attained puberty (0.72 ± 0.14 ng/μg protein) compared with gilts that did not (0.44 ± 0.05 ng/μg protein). These data provide preliminary evidence that hypothalamic expression of Kiss1 and content of kisspeptin are increased during the attainment of puberty in the pig, which may modulate the release of GnRH.