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Santana Lopez (Naya Rivera) is a competitive member of the Cheerios who joins the glee club with Quinn and Brittany in the episode "Showmance". She indicates that she and Matt are dating, but in the episode "Hairography" she 'sexts' with Puck and confronts Quinn angrily, warning her to stay away from Puck. In the episode "Sectionals" she admits that no one is forcing her to stay in glee club, adding that she likes glee and it's the best part of her day and also states that now she's taken Quinn's top dog role in school. It is also implied that she and Brittany have slept together. Santana gets her first solo lines in "The Power of Madonna", singing "Express Yourself" and "Like a Virgin". In the same episode, she sleeps with Finn Hudson after offering to take his virginity. In the episode "Laryngitis" she sings a duet of "The Boy Is Mine" with Mercedes. In "Theatricality" she gets solo lines in "Bad Romance". In "Journey", she sings lines in "Don't Stop Believin'" and, after admitting that before Glee club she hated everyone in the group, "To Sir, with Love" where she cries about the glee club being disbanded, showing that she does care.

Rivera drew on her own high school experience of unpopularity to prepare for the role, as well as watching films such as Mean Girls to "really get in the zone and feel like a bitchy Sophomore". She has described Santana as "your typical high school cheerleader, for the most part," explaining: "She's really mean and loves boys. She's really witty so I love playing her." She has characterized Santana as "a bit of a bad girl" who is "really snarky and always has these really witty one-liners she throws out there.". Rivera enjoys the fact Santana is competitive and headstrong as she herself shares those traits, but dislikes Santana's mean streak. She auditioned for the role as she loves singing, dancing and acting, and had never before had the opportunity to combine all three skills in one project. She finds the show's pacing challenging, especially the dancing, and commented in June 2009 that her most memorable moment on Glee was performing the Cheerios' glee club audition piece, "I Say a Little Prayer". Santana played a more prominent part in the last nine episodes of Glee's first season. Rivera commented: "Santana's been wreaking havoc with people's boyfriends and people's babies and teachers—she's the high school terror, and she's going to continue to be the villain."