Normal fetal testis development is essential for masculinization and subsequent adult fertility. The second trimester is a critical period of human testicular development and masculinization, but there is a paucity of reliable developmental data.The objective of the study was to analyze second-trimester human testicular morphology and function.This was an observational study of second-trimester testis development.The study was conducted at the Universities of Glasgow and Aberdeen.Testes were collected from 57 morphologically normal fetuses of women undergoing elective termination of normally progressing pregnancies (11-19 wk gestation).Testicular morphology, cell numbers, and quantitative expression of 22 key testicular genes were determined.Sertoli cell and germ cell number increased exponentially throughout the second trimester. Leydig cell number initially increased exponentially but slowed toward 19 wk. Transcripts encoding Sertoli (KITL, FGF9, SOX9, FSHR, WT1) and germ (CKIT, TFAP2C) cell-specific products increased per testis through the second trimester, but expression per cell was static apart from TFAP2C, which declined. Leydig cell transcripts (HSD17B3, CYP11A1, PTC1, CYP17, LHR, INSL3) also remained static per cell. Testicular expression of adrenal transcripts MC2R, CYP11B1, and CYP21 was detectable but unchanged. Expression of other transcripts known or postulated to be involved in testicular development (GATA4, GATA6, CXORF6, WNT2B, WNT4, WNT5A) increased significantly per testis during the second trimester.The second trimester is essential for the establishment of Sertoli and germ cell numbers. Sertoli and Leydig cells are active throughout the period, but there is no evidence of changing transcript levels.